Characterization of stress response in human retinal epithelial cells

The pathogenesis of age‐related macular degeneration (AMD) involves demise of the retinal pigment epithelium and death of photoreceptors. In this article, we investigated the response of human adult retinal pigmented epithelial (ARPE‐19) cells to 5‐(N,N‐hexamethylene)amiloride (HMA), an inhibitor of Na⁺/H⁺ exchangers. We observed that ARPE‐19 cells treated with HMA are unable to activate ‘classical’ apoptosis but they succeed to activate autophagy. In the first 2 hrs of HMA exposure, autophagy is efficient in protecting cells from death. Thereafter, autophagy is impaired, as indicated by p62 accumulation, and this protective mechanism becomes the executioner of cell death. This switch in autophagy property as a function of time for a single stimulus is here shown for the first time. The activation of autophagy was observed, at a lesser extent, with etoposide, suggesting that this event might be a general response of ARPE cells to stress and the most important pathway involved in cell resistance to adverse conditions and toxic stimuli.     

Targeting Hedgehog signaling pathway and autophagy overcomes drug resistance of BCR-ABL-positive chronic myeloid leukemia

The frontline tyrosine kinase inhibitor (TKI) imatinib has revolutionized the treatment of patients with chronic myeloid leukemia (CML). However, drug resistance is the major clinical challenge in the treatment of CML. The Hedgehog (Hh) signaling pathway and autophagy are both related to tumorigenesis, cancer therapy, and drug resistance. This study was conducted to explore whether the Hh pathway could regulate autophagy in CML cells and whether simultaneously regulating the Hh pathway and autophagy could induce cell death of drug-sensitive or -resistant BCR-ABL⁺ CML cells. Our results indicated that pharmacological or genetic inhibition of Hh pathway could markedly induce autophagy in BCR-ABL⁺ CML cells. Autophagic inhibitors or ATG5 and ATG7 silencing could significantly enhance CML cell death induced by Hh pathway suppression. Based on the above findings, our study demonstrated that simultaneously inhibiting the Hh pathway and autophagy could markedly reduce cell viability and induce apoptosis of imatinib-sensitive or -resistant BCR-ABL⁺ cells. Moreover, this combination had little cytotoxicity in human peripheral blood mononuclear cells (PBMCs). Furthermore, this combined strategy was related to PARP cleavage, CASP3 and CASP9 cleavage, and inhibition of the BCR-ABL oncoprotein. In conclusion, this study indicated that simultaneously inhibiting the Hh pathway and autophagy could potently kill imatinib-sensitive or -resistant BCR-ABL⁺ cells, providing a novel concept that simultaneously inhibiting the Hh pathway and autophagy might be a potent new strategy to overcome CML drug resistance.     

Roles of autophagy in cetuximab-mediated cancer therapy against EGFR

Cetuximab is an epidermal growth factor receptor (EGFR)-blocking antibody that is approved to treat several types of solid cancers in patients. We recently showed that cetuximab can induce autophagy in cancer cells by both inhibiting the class I phosphatidylinositol 3-kinase (PtdIns3K)/Akt/mammalian target of rapamycin (mTOR) pathway and activating the class III PtdIns3K (hVps34)/beclin 1 pathway. In the current study, we investigated the relationship between cetuximab-induced autophagy and apoptosis and the biological roles of autophagy in cetuximab-mediated cancer therapy. We found that cetuximab induced autophagy in cancer cells that show strong or weak induction of apoptosis after cetuximab treatment but not in those that show only cytostatic growth inhibition. Inhibition of cetuximab-induced apoptosis by a caspase inhibitor prevented the induction of autophagy. Conversely, inhibition of cetuximab-induced autophagy by silencing the expression of autophagy-related genes (Atg) or treating the cancer cells with lysosomal inhibitors enhanced the cetuximab-induced apoptosis, suggesting that autophagy was a protective cellular response to cetuximab treatment. On the other hand, cotreatment of cancer cells with cetuximab and the mTOR inhibitor rapamycin resulted in an Atg-dependent and lysosomal inhibition-sensitive death of cancer cells that show only growth inhibition or weak apoptosis after cetuximab treatment, indicating that cell death may be achieved by activating the autophagy pathway in these cells. Together, our findings may guide the development of novel clinical strategies for sensitizing cancer cells to EGFR-targeted therapy.Key words: EGFR, cetuximab, autophagy, apoptosis, cancer therapy     

Polygonatum odoratum lectin induces apoptosis and autophagy via targeting EGFR-mediated Ras-Raf-MEK-ERK pathway in human MCF-7 breast cancer cells

Polygonatum odoratum lectin (POL), a mannose-binding GNA-related lectin, has been reported to display remarkable anti-proliferative and apoptosis-inducing activities toward a variety of cancer cells; however, the precise molecular mechanisms by which POL induces cancer cell death are still elusive. In the current study, we found that POL could induce both apoptosis and autophagy in human MCF-7 breast cancer cells. Subsequently, we found that POL induced MCF-7 cell apoptosis via the mitochondrial pathway. Additionally, we also found that POL induces MCF-7 cell apoptosis via EGFR-mediated Ras-Raf-MEK-ERK pathway, suggesting that POL may be a potential EGFR inhibitor. Finally, we used proteomics analyses for exploring more possible POL-induced pathways with EGFR, Ras, Raf, MEK and ERK, some of which were consistent with our in silico network prediction. Taken together, these results demonstrate that POL induces MCF-7 cell apoptosis and autophagy via targeting EGFR-mediated Ras-Raf-MEK-ERK signaling pathway, which would provide a new clue for exploiting POL as a potential anti-neoplastic drug for future cancer therapy.     

Autophagy in pancreatic cancer: An emerging mechanism of cell death

Pancreatic cancer, the fourth leading cause of cancer-related death in the United States, is resistant to current chemotherapies. Therefore, identification of different pathways of cell death is important to develop novel therapeutics. Our previous study has shown that triptolide, a diterpene triepoxide, inhibits the growth of pancreatic cancer cells in vitro and prevents tumor growth in vivo. However, the mechanism by which triptolide kills pancreatic cancer cells was not known, hence, this study aimed at elucidating it. Our study reveals that triptolide kills diverse types of pancreatic cancer cells by two different pathways; it induces caspase-dependent apoptotic death in some cell lines and death via a caspase-independent autophagic pathway in the other cell lines tested. Triptolide-induced autophagy requires autophagy-specific genes, atg5 or beclin 1 and its inhibition results in cell death via the apoptotic pathway, whereas inhibition of both autophagy and apoptosis rescues triptolide-mediated cell death. Our study shows for the first time that induction of autophagy by triptolide has a pro-death role in pancreatic cancer cells. Since triptolide kills diverse pancreatic cancer cells by different mechanisms, it makes an attractive chemotherapeutic agent for future use against a broad spectrum of pancreatic cancers.Key words: pancreatic cancer, triptolide, apoptosis, caspase-3Pancreatic adenocarcinoma is one of the most lethal human malignancies. It is the fourth leading cause of cancer-related death in the United States. The five-year survival rate for pancreatic cancer is estimated to be <5% due to its aggressive growth, metastasis and resistance to radiation and most systemic chemotherapies. Hence, efforts are ongoing to understand the pathobiology of pancreatic cancer to develop innovative and effective therapies against it. A promising candidate for future therapeutic use against pancreatic cancer is a diterpene triepoxide, triptolide. Our previous studies show that triptolide inhibits the growth of pancreatic cancer cells in vitro and prevents tumor growth in vivo. Since the mechanism by which triptolide kills pancreatic cancer cells was not known, we decided to elucidate it.The K-ras, p53, p16 and DPC4 genes are the most frequently altered genes in pancreatic adenocarcinoma. In this study we have used diverse pancreatic cancer cell lines, MiaPaCa-2, Capan-1, S2-013 and S2-VP10 cells, which have mutations in all the above-mentioned genes and BxPC-3 and Hs766T cells, which have mutations in the p53, p16 and DPC4 genes, but have a wild-type K-ras gene. The treatment of all the cell lines with triptolide results in a significant time- and dose-dependent decrease in cell viability, independent of cell cycle arrest. After treatment with triptolide, only MiaPaCa-2, Capan-1 and BxPC-3 cells show an increase in the apoptosis parameters: cytochrome c release from mitochondria into the cytosol, caspase-3 activation and phosphatidylserine externalization. In contrast to this, S2-013, S2-VP10 and Hs766T cells show an induction of autophagy: an increase in LC3-II levels (by immunoblotting and immufluorescence), increase in acridine orange-positive cells, inhibition of the PtdIns3K/Akt/mTOR pathway and induction of the ERK1/2 pathway. Also, none of the cell lines tested show necrosis as evidenced by the absence of the release of lactate dehydrogenase. These results indicate that triptolide induces apoptosis in MiaPaCa-2, Capan-1 and BxPC-3 cells, whereas it induces autophagy in S2-013, S2-VP10 and Hs766T cells.Since the role of autophagy in cancer was controversial we investigated whether triptolide-induced autophagy has a prosurvival or a pro-death role. As autophagy-associated cell death is independent of caspase-3, we tested the effect of triptolide on pancreatic cancer cells in the absence of caspase-3. Treatment of cells with triptolide post-caspase-3 knockdown shows a significant rescue of cell viability only in MiaPaCa-2, but not S2-013 or S2-VP10 cells. This indicates that in contrast to MiaPaCa-2, triptolide-mediated cell death in S2-013 and S2-VP10 cells is independent of caspase-3. Next, we tested the role of autophagy in triptolide-mediated cell death in pancreatic cancer cells. In spite of a knockdown of autophagy-specific genes (atg5 and beclin 1), treatment of S2-013 and S2-VP10 cells with triptolide show a significant decline in cell viability, which is comparable to the cells treated with triptolide in the presence of autophagy genes. Subsequently we show that death in the absence of autophagy-specific genes is due to the utilization of an alternate cell death pathway, apoptosis. Furthermore, in the absence of both autophagy-specific and apoptosis-specific genes, triptolide-mediated cell death is rescued in S2-013 and S2-VP10 cells. Thus, these results confirm that triptolide-induced autophagy has a pro-death role in S2-013 and S2-VP10 cells and that these cells do not have a defect in the apoptotic machinery; however, they respond to triptolide by activating the autophagic pathway instead of the apoptotic pathway. Our studies also reveal the presence of a crosstalk between the two cell death pathways, apoptosis and autophagy, in pancreatic cancer cells.In conclusion, our study shows for the first time that triptolide induces autophagy in pancreatic cancer cells. It sheds light on the fundamental question as to whether autophagy is protective or causes cell death, proving convincingly that induction of autophagy causes cell death of some pancreatic cancer cells. Although a basal level of autophagy is necessary to maintain cellular homeostasis, its prosurvival role can be switched into a cell death mechanism if the amplitude of autophagy increases above a threshold level which is incompatible with viability, as seen in S2-013, S2-VP10 and Hs766T cells after triptolide treatment. Furthermore, there exists a crosstalk between apoptosis and autophagy in S2-013 and S2-VP10 cells; either both pathways function independently to kill the cells, with autophagy being the preferred pathway or autophagy antagonizes apoptosis and hence apoptosis is seen only after inhibiting autophagy. Although there is no direct correlation between the selection of cell death pathway in response to triptolide and the genotype of the cell lines, the choice of autophagic cell death pathway could depend on the metastatic potential of the cells; S2-013, S2-VP10 and Hs766T cell lines being more metastatic than the others, which merits further investigation. In conclusion, the ability of triptolide to induce cell death in diverse pancreatic cancer cells by either mechanism makes it an attractive chemotherapeutic agent against a broad spectrum of pancreatic cancers.     

Bufalin induces autophagy-mediated cell death in human colon cancer cells through reactive oxygen species generation and JNK activation

Colorectal cancer is the second most common cause of cancer death in the world and about half of the patients with colorectal cancer require adjuvant therapy after surgical resection. Therefore, the eradication of cancer cells via chemotherapy constitutes a viable approach to treating patients with colorectal cancer. In this study, the effects of bufalin isolated from a traditional Chinese medicine were evaluated and characterized in HT-29 and Caco-2 human colon cancer cells. Contrary to its well-documented apoptosis-promoting activity in other cancer cells, bufalin did not cause caspase-dependent cell death in colon cancer cells, as indicated by the absence of significant early apoptosis as well as poly(ADP-ribose) polymerase and caspase-3 cleavage. Instead, bufalin activated an autophagy pathway, as characterized by the accumulation of LC3-II and the stimulation of autophagic flux. The induction of autophagy by bufalin was linked to the generation of reactive oxygen species (ROS). ROS activated autophagy via the c-Jun NH₂-terminal kinase (JNK). JNK activation increased expression of ATG5 and Beclin-1. ROS antioxidants (N-acetylcysteine and vitamin C), the JNK-specific inhibitor SP600125, and JNK2 siRNA attenuated bufalin-induced autophagy. Our findings unveil a novel mechanism of drug action by bufalin in colon cancer cells and open up the possibility of treating colorectal cancer through a ROS-dependent autophagy pathway.     

QSOX1 Inhibits Autophagic Flux in Breast Cancer Cells

The QSOX1 protein (Quiescin Sulfhydryl oxidase 1) catalyzes the formation of disulfide bonds and is involved in the folding and stability of proteins. More recently, QSOX1 has been associated with tumorigenesis and protection against cellular stress. It has been demonstrated in our laboratory that QSOX1 reduces proliferation, migration and invasion of breast cancer cells in vitro and reduces tumor growth in vivo. In addition, QSOX1 expression has been shown to be induced by oxidative or ER stress and to prevent cell death linked to these stressors. Given the function of QSOX1 in these two processes, which have been previously linked to autophagy, we wondered whether QSOX1 might be regulated by autophagy inducers and play a role in this catabolic process. To answer this question, we used in vitro models of breast cancer cells in which QSOX1 was overexpressed (MCF-7) or extinguished (MDA-MB-231). We first showed that QSOX1 expression is induced following amino acid starvation and maintains cellular homeostasis. Our results also indicated that QSOX1 inhibits autophagy through the inhibition of autophagosome/lysosome fusion. Moreover, we demonstrated that inhibitors of autophagy mimic the effect of QSOX1 on cell invasion, suggesting that its role in this process is linked to the autophagy pathway. Previously published data demonstrated that extinction of QSOX1 promotes tumor growth in NOG mice. In this study, we further demonstrated that QSOX1 null tumors present lower levels of the p62 protein. Altogether, our results demonstrate for the first time a role of QSOX1 in autophagy in breast cancer cells and tumors.     

Piperlongumine induces autophagy by targeting p38 signaling

Piperlongumine (PL), a natural product isolated from the plant species Piper longum L., can selectively induce apoptotic cell death in cancer cells by targeting the stress response to reactive oxygen species (ROS). Here we show that PL induces cell death in the presence of benzyloxycarbonylvalyl-alanyl-aspartic acid (O-methyl)-fluoro-methylketone (zVAD-fmk), a pan-apoptotic inhibitor, and in the presence of necrostatin-1, a necrotic inhibitor. Instead PL-induced cell death can be suppressed by 3-methyladenine, an autophagy inhibitor, and substantially attenuated in cells lacking the autophagy-related 5 (Atg5) gene. We further show that PL enhances autophagy activity without blocking autophagy flux. Application of N-acetyl-cysteine, an antioxidant, markedly reduces PL-induced autophagy and cell death, suggesting an essential role for intracellular ROS in PL-induced autophagy. Furthermore, PL stimulates the activation of p38 protein kinase through ROS-induced stress response and p38 signaling is necessary for the action of PL as SB203580, a p38 inhibitor, or dominant-negative p38 can effectively reduce PL-mediated autophagy. Thus, we have characterized a new mechanism for PL-induced cell death through the ROS-p38 pathway. Our findings support the therapeutic potential of PL by triggering autophagic cell death.     

Targeting Hedgehog signaling pathway and autophagy overcomes drug resistance of BCR-ABL-positive chronic myeloid leukemia

The frontline tyrosine kinase inhibitor (TKI) imatinib has revolutionized the treatment of patients with chronic myeloid leukemia (CML). However, drug resistance is the major clinical challenge in the treatment of CML. The Hedgehog (Hh) signaling pathway and autophagy are both related to tumorigenesis, cancer therapy, and drug resistance. This study was conducted to explore whether the Hh pathway could regulate autophagy in CML cells and whether simultaneously regulating the Hh pathway and autophagy could induce cell death of drug-sensitive or -resistant BCR-ABL⁺ CML cells. Our results indicated that pharmacological or genetic inhibition of Hh pathway could markedly induce autophagy in BCR-ABL⁺ CML cells. Autophagic inhibitors or ATG5 and ATG7 silencing could significantly enhance CML cell death induced by Hh pathway suppression. Based on the above findings, our study demonstrated that simultaneously inhibiting the Hh pathway and autophagy could markedly reduce cell viability and induce apoptosis of imatinib-sensitive or -resistant BCR-ABL⁺ cells. Moreover, this combination had little cytotoxicity in human peripheral blood mononuclear cells (PBMCs). Furthermore, this combined strategy was related to PARP cleavage, CASP3 and CASP9 cleavage, and inhibition of the BCR-ABL oncoprotein. In conclusion, this study indicated that simultaneously inhibiting the Hh pathway and autophagy could potently kill imatinib-sensitive or -resistant BCR-ABL⁺ cells, providing a novel concept that simultaneously inhibiting the Hh pathway and autophagy might be a potent new strategy to overcome CML drug resistance.     

Ursolic acid induces cell death and modulates autophagy through JNK pathway in apoptosis-resistant colorectal cancer cells

Colorectal carcinomas (CRCs) with P53 mutations have been shown to be resistant to chemotherapy with 5-fluorouracil (5-FU), the most widely used chemotherapeutic drug for CRC treatment. Autophagy is emerging as a promising therapeutic target for drug-resistant tumors. In the present study, we tested the effects of ursolic acid (UA), a natural triterpenoid, on cell death mechanisms and its effects in combination with 5-FU in the HCT15 p53 mutant apoptosis-resistant CRC cell line. The involvement of UA in autophagy and its in vivo efficacy were evaluated.Our data show that UA induces apoptosis independent of caspases in HCT15 cells and enhances 5-FU effects associated with an activation of c-jun N-terminal kinase (JNK). In this cell line, where this compound has a more pronounced effect on the induction of cell death compared to 5-FU, apoptosis corresponds only to a small percentage of the total cell death induced by UA. UA also modulated autophagy by inducing the accumulation of LC3 and p62 levels with involvement of JNK pathway, which indicates a contribution of autophagy on JNK-dependent induction of cell death by UA. By using nude mice xenografted with HCT15 cells, we verified that UA was also active in vivo decreasing tumor growth rate.In conclusion, this study shows UA's anticancer potential both in vitro and in vivo. Induction of cell death and modulation of autophagy in CRC-resistant cells were shown to involve JNK signaling.     

Autophagy is not uniformly cytoprotective: a personalized medicine approach for autophagy inhibition as a therapeutic strategy in non-small cell lung cancer

BackgroundLung cancer is the leading cause of cancer-related death worldwide. In addition to surgical resection, which is considered first-line treatment at early stages of the disease, chemotherapy and radiation are widely used when the disease is advanced. Of multiple responses that may occur in the tumor cells in response to cancer therapy, the functional importance of autophagy remains equivocal; this is likely to restrict current efforts to sensitize this malignancy to chemotherapy and/or radiation by pharmacological interference with the autophagic response.Scope of reviewIn this review, we attempt to summarize the current state of knowledge based on studies that evaluated the function of autophagy in non-small cell lung cancer (NSCLC) cells in response to radiation and the most commonly used chemotherapeutic agents.Major conclusionsIn addition to the expected prosurvival function of autophagy, where autophagy inhibition enhances the response to therapy, autophagy appears also to have a “non-cytoprotective” function, where autophagy blockade does not affect cell viability, clonogenicity or tumor volume in response to therapy. In other cases, autophagy may actually mediate drug action via expression of its cytotoxic function.General significanceThese observations emphasize the complexity of autophagy function when examined in different tumor cell lines and in response to different chemotherapeutic agents. A more in-depth understanding of the conditions that promote the unique functions of autophagy is required in order to translate preclinical findings of autophagy inhibition to the clinic for the purpose of improving patient response to chemotherapy and radiation.     

Autophagy is a therapeutic target in anticancer drug resistance

Autophagy is a type of cellular catabolic degradation response to nutrient starvation or metabolic stress. The main function of autophagy is to maintain intracellular metabolic homeostasis through degradation of unfolded or aggregated proteins and organelles. Although autophagic regulation is a complicated process, solid evidence demonstrates that the PI3K-Akt-mTOR, LKB1-AMPK-mTOR and p53 are the main upstream regulators of the autophagic pathway. Currently, there is a bulk of data indicating the important function of autophagy in cancer. It is noteworthy that autophagy facilitates the cancer cells' resistance to chemotherapy and radiation treatment. The abrogation of autophagy potentiates the re-sensitization of therapeutic resistant cancer cells to the anticancer treatment via autophagy inhibitors, such as 3-MA, CQ and BA, or knockdown of the autophagy related molecules. In this review, we summarize the accumulation of evidence for autophagy's involvement in mediating resistance of cancer cells to anticancer therapy and suggest that autophagy might be a potential therapeutic target in anticancer drug resistance in the future.     

Inhibition of autophagy by 3-MA potentiates purvalanol-induced apoptosis in Bax deficient HCT 116 colon cancer cells

The purine-derived analogs, roscovitine and purvalanol are selective synthetic inhibitors of cyclin-dependent kinases (CDKs) induced cell cycle arrest and lead to apoptotic cell death in various cancer cells. Although a number of studies investigated the molecular mechanism of each CDK inhibitor on apoptotic cell death mechanism with their therapeutic potential, their regulatory role on autophagy is not clarified yet. In this paper, our aim was to investigate molecular mechanism of CDK inhibitors on autophagy and apoptosis in wild type (wt) and Bax deficient HCT 116 cells. Exposure of HCT 116 wt and Bax^−/− cells to roscovitine or purvalanol for 24 h decreased cell viability in dose-dependent manner. However, Bax deficient HCT 116 cells were found more resistant against purvalanol treatment compared to wt cells. We also established that both CDK inhibitors induced apoptosis through activating mitochondria-mediated pathway in caspase-dependent manner regardless of Bax expression in HCT 116 colon cancer cells. Concomitantly, we determined that purvalanol was also effective on autophagy in HCT 116 colon cancer cells. Inhibition of autophagy by 3-MA treatment enhanced the purvalanol induced apoptotic cell death in HCT 116 Bax^−/− cells. Our results revealed that mechanistic action of each CDK inhibitor on cell death mechanism differs. While purvalanol treatment activated apoptosis and autophagy in HCT 116 cells, roscovitine was only effective on caspase-dependent apoptotic pathway. Another important difference between two CDK inhibitors, although roscovitine treatment overcame Bax-mediated drug resistance in HCT 116 cells, purvalanol did not exert same effect.     

TOR-mediated autophagy regulates cell death in Drosophila neurodegenerative disease

Target of rapamycin (TOR) signaling is a regulator of cell growth. TOR activity can also enhance cell death, and the TOR inhibitor rapamycin protects cells against proapoptotic stimuli. Autophagy, which can protect against cell death, is negatively regulated by TOR, and disruption of autophagy by mutation of Atg5 or Atg7 can lead to neurodegeneration. However, the implied functional connection between TOR signaling, autophagy, and cell death or degeneration has not been rigorously tested. Using the Drosophila melanogaster visual system, we show in this study that hyperactivation of TOR leads to photoreceptor cell death in an age- and light-dependent manner and that this is because of TOR''s ability to suppress autophagy. We also find that genetically inhibiting TOR or inducing autophagy suppresses cell death in Drosophila models of Huntington''s disease and phospholipase C (norpA)–mediated retinal degeneration. Thus, our data indicate that TOR induces cell death by suppressing autophagy and provide direct genetic evidence that autophagy alleviates cell death in several common types of neurodegenerative disease.     

Apoptotic-like Leishmania exploit the host′s autophagy machinery to reduce T-cell-mediated parasite elimination

Apoptosis is a well-defined cellular process in which a cell dies, characterized by cell shrinkage and DNA fragmentation. In parasites like Leishmania, the process of apoptosis-like cell death has been described. Moreover upon infection, the apoptotic-like population is essential for disease development, in part by silencing host phagocytes. Nevertheless, the exact mechanism of how apoptosis in unicellular organisms may support infectivity remains unclear. Therefore we investigated the fate of apoptotic-like Leishmania parasites in human host macrophages. Our data showed—in contrast to viable parasites—that apoptotic-like parasites enter an LC3⁺, autophagy-like compartment. The compartment was found to consist of a single lipid bilayer, typical for LC3-associated phagocytosis (LAP). As LAP can provoke anti-inflammatory responses and autophagy modulates antigen presentation, we analyzed how the presence of apoptotic-like parasites affected the adaptive immune response. Macrophages infected with viable Leishmania induced proliferation of CD4⁺ T-cells, leading to a reduced intracellular parasite survival. Remarkably, the presence of apoptotic-like parasites in the inoculum significantly reduced T-cell proliferation. Chemical induction of autophagy in human monocyte-derived macrophage (hMDM), infected with viable parasites only, had an even stronger proliferation-reducing effect, indicating that host cell autophagy and not parasite viability limits the T-cell response and enhances parasite survival. Concluding, our data suggest that apoptotic-like Leishmania hijack the host cells´ autophagy machinery to reduce T-cell proliferation. Furthermore, the overall population survival is guaranteed, explaining the benefit of apoptosis-like cell death in a single-celled parasite and defining the host autophagy pathway as a potential therapeutic target in treating Leishmaniasis.     

Polygonatum cyrtonema lectin induces murine fibrosarcoma L929 cell apoptosis and autophagy via blocking Ras–Raf and PI3K–Akt signaling pathways

Polygonatum cyrtonema lectin (PCL), a mannose/sialic acid-binding lectin, has been reported to display remarkable anti-proliferative and apoptosis-inducing activities toward a variety of cancer cells; however, the precise molecular mechanisms by which PCL induces cancer cell death are still elusive. In the current study, we found that PCL could induce apoptosis and autophagy in murine fibrosarcoma L929 cells. Subsequently, we demonstrated that inhibition of Ras could promote L929 cell death, suggesting that Ras–Raf signaling pathway plays the key negative regulator in PCL-induced apoptosis. And, we showed that Ras-Raf signaling pathway was also involved in PCL-induced autophagy as the negative regulator. In addition, we found that class I phosphatidylinositol 3-kinase (PI3K)–Akt signaling pathway could play the negative regulator in PCL-induced apoptosis and autophagy. Taken together, these results demonstrate that PCL induces murine fibrosarcoma L929 cell apoptosis and autophagy via blocking Ras-Raf and PI3K–Akt signaling pathways.     

The variability of autophagy and cell death susceptibility

Impaired autophagic machinery is implicated in a number of diseases such as heart disease, neurodegeneration and cancer. A common denominator in these pathologies is a dysregulation of autophagy that has been linked to a change in susceptibility to cell death. Although we have progressed in understanding the molecular machinery and regulation of the autophagic pathway, many unanswered questions remain. How does the metabolic contribution of autophagy connect with the cell’s history and how does its current autophagic flux affect metabolic status and susceptibility to undergo cell death? How does autophagic flux operate to switch metabolic direction and what are the underlying mechanisms in metabolite and energetic sensing, metabolite substrate provision and metabolic integration during the cellular stress response? In this article we focus on unresolved questions that address issues around the role of autophagy in sensing the energetic environment and its role in actively generating metabolite substrates. We attempt to provide answers by explaining how and when a change in autophagic pathway activity such as primary stress response is able to affect cell viability and when not. By addressing the dynamic metabolic relationship between autophagy, apoptosis and necrosis we provide a new perspective on the parameters that connect autophagic activity, severity of injury and cellular history in a logical manner. Last, by evaluating the cell’s condition and autophagic activity in a clear context of regulatory parameters in the intra- and extracellular environment, this review provides new concepts that set autophagy into an energetic feedback loop, that may assist in our understanding of autophagy in maintaining healthy cells or when it controls the threshold between cell death and cell survival.     

Autophagy triggered by magnolol derivative negatively regulates angiogenesis

Angiogenesis has a key role in the tumor progression and metastasis; targeting endothelial cell proliferation has emerged as a promising therapeutic strategy for the prevention of cancer. Previous studies have revealed a complex association between the process of angiogenesis and autophagy and its outcome on tumorigenesis. Autophagy, also known as type-II cell death, has been identified as an alternative way of cell killing in apoptotic-resistant cancer cells. However, its involvement in chemoresistance and tumor promotion is also well known. In this study, we used a derivate of natural product magnolol (Ery5), a potent autophagy inducer, to study the association between the autophagy and angiogenesis in both in vitro and in vivo model system. We found that the robust autophagy triggered by Ery5, inhibited angiogenesis and caused cell death independent of the apoptosis in human umbilical cord vein endothelial cells and PC-3 cells. Ery5 induced autophagy effectively inhibited cell proliferation, migration, invasion and tube formation. We further demonstrated that Ery5-mediated autophagy and subsequent inhibition of angiogenesis was reversed when autophagy was inhibited through 3-methyl adenine and knocking down of key autophagy proteins ATG7 and microtubule-associated protein light chain 3. While evaluating the negative regulation of autophagy on angiogenesis, it was interesting to find that angiogenic environment produced by the treatment of VEGF and CoCl2 remarkably downregulated the autophagy and autophagic cell death induced by Ery5. These studies, while disclosing the vital role of autophagy in the regulation of angiogenesis, also suggest that the potent modulators of autophagy can lead to the development of effective therapeutics in apoptosis-resistant cancer.