Oxyresveratrol activates parallel apoptotic and autophagic cell death pathways in neuroblastoma cells

BackgroundDrug resistance from apoptosis is a challenging issue with different cancer types, and there is an interest in identifying other means of inducing cytotoxicity. Here, treatment of neuroblastoma cells with oxyresveratrol (OXYRES), a natural antioxidant, led to dose-dependent cell death and increased autophagic flux along with activation of caspase-dependent apoptosis.MethodsFor cell viability, we performed the CCK-8 assay. Protein expression changes were with Western blot and immunocytochemistry. Silencing of proteins was with siRNA. The readouts for cell cycle, mitochondria membrane potential, caspase-3, autophagy and apoptosis were performed with flow cytometry.ResultsPhosphorylation of p38 MAPK increased with OXYRES treatment and inhibition of p38 reduced autophagy and cell death from OXYRES. In contrast, PI3K/AKT/mTOR signaling decreased in the target cells with OXYRES and inhibition of PI3K or mTOR enhanced OXYRES-mediated cytotoxicity with increased levels of autophagy. Modulation of either of the apoptosis and autophagy flux pathways affected the extent of cell death by OXYRES, but did not affect the indicators of these pathways with respect to each other. Both pathways were independent of ROS generation or p53 activation.ConclusionOXYRES led to cell death from autophagy, which was independent of apoptosis induction. The OXYRES effects were due to changes in the activity levels of p38 MAPK and PI3K/AKT/mTOR.General significanceWith two independent and parallel pathways for cytotoxicity induction in target cells, this study puts forward a potential utility for OXYRES or the pathways it represents as novel means of inducing cell death in neuroblastoma cells.     

mTOR is a fine tuning molecule in CDK inhibitors-induced distinct cell death mechanisms via PI3K/AKT/mTOR signaling axis in prostate cancer cells

Purvalanol and roscovitine are cyclin dependent kinase (CDK) inhibitors that induce cell cycle arrest and apoptosis in various cancer cells. We further hypothesized that co-treatment of CDK inhibitors with rapamycin, an mTOR inhibitor, would be an effective combinatory strategy for the inhibition of prostate cancer regard to androgen receptor (AR) status due to inhibition of proliferative pathway, PI3K/AKT/mTOR, and induction of cell death mechanisms. Androgen responsive (AR+), PTEN^?/? LNCaP and androgen independent (AR?), PTEN^+/? DU145 prostate cancer cells were exposed to purvalanol (20 µM) and roscovitine (30 µM) with or without rapamycin for 24 h. Cell viability assay, immunoblotting, flow cytometry and fluorescence microscopy was used to define the effect of CDK inhibitors with or without rapamycin on proliferative pathway and cell death mechanisms in LNCaP and DU145 prostate cancer cells. Co-treatment of rapamycin modulated CDK inhibitors-induced cytotoxicity and apoptosis that CDK inhibitors were more potent to induce cell death in AR (+) LNCaP cells than AR (?) DU145 cells. CDK inhibitors in the presence or absence of rapamycin induced cell death via modulating upstream PI3K/AKT/mTOR signaling pathway in LNCaP cells, exclusively only treatment of purvalanol have strong potential to inhibit both upstream and downstream targets of mTOR in LNCaP and DU145 cells. However, co-treatment of rapamycin with CDK inhibitors protects DU145 cells from apoptosis via induction of autophagy mechanism. We confirmed that purvalanol and roscovitine were strong apoptotic and autophagy inducers that based on regulation of PI3K/AKT/mTOR signaling pathway. Co-treatment of rapamycin with purvalanol and roscovitine exerted different effects on cell survival and death mechanisms in LNCaP and DU145 cell due to their AR receptor status. Our studies show that co-treatment of rapamycin with CDK inhibitors inhibit prostate cancer cell viability more effectively than either agent alone, in part, by targeting the mTOR signaling cascade in AR (+) LNCaP cells. In this point, mTOR is a fine-tuning player in purvalanol and roscovitine-induced apoptosis and autophagy via regulation of PI3K/AKT and the downstream targets, which related with cell proliferation.     

Mollugin induces tumor cell apoptosis and autophagy via the PI3K/AKT/mTOR/p70S6K and ERK signaling pathways

Mollugin, a bioactive phytochemical isolated from Rubia cordifolia L., has shown preclinical anticancer efficacy in various cancer models. However the effects of mollugin in regulating cancer cell survival and death remains undefined. In the present study we found that mollugin exhibited cytotoxicity on various cancer models. The suppression of cell viability was due to the induction of mitochondria apoptosis. In addition, the presence of autophagic hallmarks was observed in mollugin-treated cells. Notably, blockade of autophagy by a chemical inhibitor or RNA interference enhanced the cytotoxicity of mollugin. Further experiments demonstrated that phosphatidylinositide 3-kinases/protein kinase B/mammalian target of rapamycin/p70S6 kinase (PI3K/AKT/mTOR/p70S6K) and extracellular regulated protein kinases (ERK) signaling pathways participated in mollugin-induced autophagy and apoptosis. Together, these findings support further studies of mollugin as candidate for treatment of human cancer cells.     

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.     

Direct induction of autophagy by Atg1 inhibits cell growth and induces apoptotic cell death

BACKGROUND: To survive starvation and other forms of stress, eukaryotic cells undergo a lysosomal process of cytoplasmic degradation known as autophagy. Autophagy has been implicated in a number of cellular and developmental processes, including cell-growth control and programmed cell death. However, direct evidence of a causal role for autophagy in these processes is lacking, resulting in part from the pleiotropic effects of signaling molecules such as TOR that regulate autophagy. Here, we circumvent this difficulty by directly manipulating autophagy rates in Drosophila through the autophagy-specific protein kinase Atg1. RESULTS: We find that overexpression of Atg1 is sufficient to induce high levels of autophagy, the first such demonstration among wild-type Atg proteins. In contrast to findings in yeast, induction of autophagy by Atg1 is dependent on its kinase activity. We find that cells with high levels of Atg1-induced autophagy are rapidly eliminated, demonstrating that autophagy is capable of inducing cell death. However, this cell death is caspase dependent and displays DNA fragmentation, suggesting that autophagy represents an alternative induction of apoptosis, rather than a distinct form of cell death. In addition, we demonstrate that Atg1-induced autophagy strongly inhibits cell growth and that Atg1 mutant cells have a relative growth advantage under conditions of reduced TOR signaling. Finally, we show that Atg1 expression results in negative feedback on the activity of TOR itself. CONCLUSIONS: Our results reveal a central role for Atg1 in mounting a coordinated autophagic response and demonstrate that autophagy has the capacity to induce cell death. Furthermore, this work identifies autophagy as a critical mechanism by which inhibition of TOR signaling leads to reduced cell growth.     

An Atg1/Atg13 Complex with Multiple Roles in TOR-mediated Autophagy Regulation

The TOR kinases are conserved negative regulators of autophagy in response to nutrient conditions, but the signaling mechanisms are poorly understood. Here we describe a complex containing the protein kinase Atg1 and the phosphoprotein Atg13 that functions as a critical component of this regulation in Drosophila. We show that knockout of Atg1 or Atg13 results in a similar, selective defect in autophagy in response to TOR inactivation. Atg1 physically interacts with TOR and Atg13 in vivo, and both Atg1 and Atg13 are phosphorylated in a nutrient-, TOR- and Atg1 kinase-dependent manner. In contrast to yeast, phosphorylation of Atg13 is greatest under autophagic conditions and does not preclude Atg1-Atg13 association. Atg13 stimulates both the autophagic activity of Atg1 and its inhibition of cell growth and TOR signaling, in part by disrupting the normal trafficking of TOR. In contrast to the effects of normal Atg13 levels, increased expression of Atg13 inhibits autophagosome expansion and recruitment of Atg8/LC3, potentially by decreasing the stability of Atg1 and facilitating its inhibitory phosphorylation by TOR. Atg1-Atg13 complexes thus function at multiple levels to mediate and adjust nutrient-dependent autophagic signaling.     

ROS accumulation contributes to abamectin‐induced apoptosis and autophagy via the inactivation of PI3K/AKT/mTOR pathway in TM3 Leydig cells

Abamectin (ABA) as one of the worldwide used compounds in agriculture has raised safety concerns on nontarget organism toxicity. However, the study of male reproductive system damage caused by ABA remains unclear. Our aim is to investigate the effect of ABA‐induced cytotoxicity in TM3 Leydig cells and their underlying mechanisms. ABA inhibits TM3 cell viability and proliferation via cell cycle arrested in the G0/G1 phase. In addition, ABA‐induced mitochondrial depolarization leads to an imbalance in Bcl‐2 family expression, causing caspase‐dependent apoptosis in TM3 cells. The increased ratio of cells expression LC3 protein and LC3‐II to LC3‐I indicated the activation of autophagy potentially. Further experiments revealed ABA treatment reduced phosphatidylinositol 3‐kinase (PI3K), protein kinase B (AKT) phosphorylation, and mammalian target of rapamycin (mTOR) phosphorylation. Pretreatment with a PI3K/AKT inhibitor, LY294002, mimicked the ABA‐mediated effects on cytotoxicity. Pretreatment with a PI3K/AKT agonist, insulin‐like growth factor‐1, reversed the effects of ABA. ABA caused the accumulation of intracellular reactive oxygen species (ROS) by increased intensity of the ROS indicator. However, N‐acetylcysteine as ROS scavengers inhibited ABA‐induced apoptosis and autophagy and reversed these ABA‐mediated effects on PI3K/AKT/mTOR pathway. On the basis of the above results, it is suggested that ABA exposure induces apoptosis and autophagy in TM3 cells by ROS accumulation to mediate PI3K/AKT/mTOR signaling pathway suppression.     

Autophagy mediates cytotoxicity of human colorectal cancer cells treated with garcinielliptone FC

The tautomeric pair of garcinielliptone FC (GFC) is a novel tautomeric pair of polyprenyl benzophenonoid isolated from the pericarps of Garcinia subelliptica Merr. (G. subelliptica, Clusiaceae), a tree with abundant sources of polyphenols. Our previous report demonstrated that GFC induced apoptosis on various types of human cancer cell lines including chemoresistant human colorectal cancer HT‐29 cells. In the present study, we observed that many autophagy‐related genes in GFC‐treated HT‐29 cells were up‐ and down‐regulated using a cDNA microarray containing oncogenes and kinase genes. GFC‐induced autophagy of HT‐29 cells was confirmed by observing the formation of acidic vesicular organelles, LC3 puncta, and double‐membrane autophagic vesicles using flow cytometry, confocal microscopy, and transmission electron microscopy, respectively. Inhibition of AKT/mTOR/P70S6K signaling as well as formation of Atg5‐Atg12 and PI3K/Beclin‐1 complexes were observed using Western blot. Administration of autophagy inhibitor (3‐methyladenine and shRNA Atg5) and apoptosis inhibitor Z‐VAD showed that the GFC‐induced autophagy was cytotoxic form and GFC‐induced apoptosis enhanced GFC‐induced autophagy. Our data suggest the involvement of autophagy and apoptosis in GFC‐induced anticancer mechanisms of human colorectal cancer.     

Dual inhibition of BDNF/TrkB and autophagy: a promising therapeutic approach for colorectal cancer

Colorectal cancer (CRC) is the most common digestive cancer in the Western world. Despite effective therapies, resistance and/or recurrence frequently occur. The present study investigated the impact of two survival pathways—neurotrophic factors (TrkB/BDNF) and autophagy—on cell fate and tumour evolution. In vitro studies were performed on two CRC cell lines, SW480 (primary tumour) and SW620 (lymph node invasion), which were also used for subcutaneous xenografts on a nude mouse model. In addition, the presence of neurotrophic factors (NTs) and autophagy markers were assessed in tissue samples representative of different stages. On the basis of our previous study (which demonstrated that TrkB overexpression is associated with prosurvival signaling in CRC cells), we pharmacologically inhibited NTs pathways with K252a. As expected, an inactivation of the PI3K/AKT pathway was observed and CRC cells initiated autophagy. Conversely, blocking the autophagic flux with chloroquine or with ATG5‐siRNA overactivated TrkB/BDNF signaling. In vitro, dual inhibition improved the effectiveness of single treatment by significantly reducing metabolic activity and enhancing apoptotic cell death. These findings were accentuated in vivo, in which dual inhibition induced a spectacular reduction in tumour volume following long‐term treatment (21 days for K252a and 12 days for CQ). Finally, significant amounts of phospho‐TrkB and LC3II were found in the patients’ tissues, highlighting their relevance in CRC tumour biology. Taken together, our results show that targeting NTs and autophagy pathways potentially constitutes a new therapeutic approach for CRC.     

ERK-mediated autophagy promotes inactivated Sendai virus (HVJ-E)-induced apoptosis in HeLa cells in an Atg3-dependent manner

Background

Apoptosis and autophagy are known to play important roles in cancer development. It has been reported that HVJ-E induces apoptosis in cancer cells, thereby inhibiting the development of tumors. To define the mechanism by which HVJ-E induces cell death, we examined whether HVJ-E activates autophagic and apoptotic signaling pathways in HeLa cells.

Methods

Cells were treated with chloroquine (CQ) and rapamycin to determine whether autophagy is involved in HVJ-E-induced apoptosis. Treatment with the ERK inhibitor, U0126, was used to determine whether autophagy and apoptosis are mediated by the ERK pathway. Activators of the PI3K/Akt/mTOR/p70S6K pathway, 740 Y-P and SC79, were used to characterize its role in HVJ-E-induced autophagy. siRNA against Atg3 was used to knock down the protein and determine whether it plays a role in HVJ-E-induced apoptosis in HeLa cells.

Results

We found that HVJ-E infection inhibited cell viability and induced apoptosis through the mitochondrial pathway, as evidenced by the expression of caspase proteins. This process was promoted by rapamycin treatment and inhibited by CQ treatment. HVJ-E-induced autophagy was further blocked by 740 Y-P, SC79, and U0126, indicating that both the ERK- and the PI3K/Akt/mTOR/p70S6K-pathways were involved. Finally, autophagy-mediated apoptosis induced by HVJ-E was inhibited by siRNA-mediated Atg3 knockdown.

Conclusion

In HeLa cells, HVJ-E infection triggered autophagy through the PI3K/Akt/mTOR/p70S6K pathway in an ERK1/2-dependent manner, and the induction of autophagy promoted apoptosis in an Atg3-dependent manner.

     

Crosstalk between Raf/MEK/ERK and PI3K/AKT in suppression of Bax conformational change by Grp75 under glucose deprivation conditions

During glucose deprivation (GD)-induced cellular stress, the molecular chaperone glucose-regulated protein 75 (Grp75)/Mortalin/PBP74/mtHSP70 (hereafter termed “Grp75”) plays an important role in the suppression of apoptosis by inhibiting the Bax conformational change that delays the release of cytochrome c. The molecular pathways by which it carries out these functions are still unclear. We hypothesize that the anti-apoptotic effect by the overexpression of Grp75 was through the signal of AKT activated by classic phosphoinositide 3-kinase (PI3K) and also involved PI3K-independent pathways. Using the PC12 cell GD model, we demonstrated a novel mechanism of Grp75 activating AKT, which may be PI3K independent and associated with Raf/MEK (mitogen-activated protein kinase/ERK kinase)/ERK signaling. The PI3K inhibitor LY294002 did not influence the activation of AKT by the Grp75 overexpression under GD; however, the MEK inhibitor U0126 dramatically inhibited AKT phosphorylation in the same assay. In addition to the PI3K/AKT signal pathway, Grp75 overexpression also inhibited the Bax conformational change through the Raf/MEK/ERK signal pathway. In conclusion, Grp75 overexpression in activating AKT can be PI3K independent and associated with Raf/MEK/ERK signaling under GD. At the same time, PI3K may also crosstalk with Raf-1, in which the prosurvival signal of PI3K maintains the expression of Raf-1. The activated AKT and extracellular signal-regulated protein kinases 1 and 2 by Grp75 inhibited the Bax conformational change and subsequent apoptosis.     

Contribution of Atg1-Dependent Autophagy to TOR-Mediated Cell Growth and Survival

The Ser/Thr kinase Atg1 (Ulk1/Unc51) appears to act as a convergence point for multiple signals that regulate autophagy, and in turn interacts with a large number of autophagy-related (Atg) proteins. Working in the Drosophila system, we recently found that overexpression of Atg1 is sufficient to induce autophagy, independent of upstream nutrient signals. We exploited this finding to examine the roles of autophagy in cell growth and death, and to test the interaction of Atg1 with the TOR signaling pathway. These studies provided genetic evidence that autophagy is a potent inhibitor of cell growth, and that high levels of autophagy lead to caspase-dependent apoptotic cell death in vivo. Atg1 also has an inhibitory effect on TOR signaling, indicating the existence of a positive feedback mechanism that may amplify the nutrient-dependent signals that control autophagy.

Addendum to:

Direct Induction of Autophagy by Atg1 Inhibits Cell Growth and Induces Apoptotic Cell Death

R.C. Scott, G. Juhász and T.P. Neufeld

Curr Biol 2007; 17:1-11     

Contribution of Atg1-dependent autophagy to TOR-mediated cell growth and survival

The Ser/Thr kinase Atg1 (Ulk1/Unc51) appears to act as a convergence point for multiple signals that regulate autophagy, and in turn interacts with a large number of autophagy-related (Atg) proteins. Working in the Drosophila system, we recently found that overexpression of Atg1 is sufficient to induce autophagy, independent of upstream nutrient signals. We exploited this finding to examine the roles of autophagy in cell growth and death, and to test the interaction of Atg1 with the TOR signaling pathway. These studies provided genetic evidence that autophagy is a potent inhibitor of cell growth, and that high levels of autophagy lead to caspase-dependent apoptotic cell death in vivo. Atg1 also has an inhibitory effect on TOR signaling, indicating the existence of a positive feedback mechanism that may amplify the nutrient-dependent signals that control autophagy.     

Concomitant reduction of c-Myc expression and PI3K/AKT/mTOR signaling by quercetin induces a strong cytotoxic effect against Burkitt’s lymphoma

Burkitt’s lymphoma is an aggressive B cell lymphoma whose pathogenesis involves mainly c-Myc translocation and hyperexpression, in addition to antigen-independent BCR signaling and, in some cases, EBV infection. As result of BCR signaling activation, the PI3K/AKT/mTOR pathway results constitutively activated also in the absence of EBV, promoting cell survival and counterbalancing the pro-apoptotic function that c-Myc may also exert. In this study we found that quercetin, a bioflavonoid widely distributed in plant kingdom, reduced c-Myc expression and inhibited the PI3K/AKT/mTOR activity in BL, leading to an apoptotic cell death. We observed a higher cytotoxic effect against the EBV-negative BL cells in comparison with the positive ones, suggesting that this oncogenic gammaherpesvirus confers an additional resistance to the quercetin treatment. Besides cell survival, PI3K/AKT/mTOR pathway also regulates autophagy: we found that quercetin induced a complete autophagic flux in BL cells, that contributes to c-Myc reduction in some of these cells. Indeed, autophagy inhibition by chloroquine partially restored c-Myc expression in EBV-positive (Akata) and EBV-negative (2A8) cells that harbor c-Myc mutation. Interestingly, chloroquine did not affect the quercetin-mediated reduction of c-Myc expression in Ramos cells, that have no c-Myc mutation in the coding region, although autophagy was induced.These results suggest that mutant c-Myc could be partially degraded through autophagy in BL cells, as previously reported for other mutant oncogenic proteins.     

PIK3CA‐mutated melanoma cells rely on cooperative signaling through mTORC1/2 for sustained proliferation

Malignant conversion of BRAF‐ or NRAS‐mutated melanocytes into melanoma cells can be promoted by PI3′‐lipid signaling. However, the mechanism by which PI3′‐lipid signaling cooperates with mutationally activated BRAF or NRAS has not been adequately explored. Using human NRAS‐ or BRAF‐mutated melanoma cells that co‐express mutationally activated PIK3CA, we explored the contribution of PI3′‐lipid signaling to cell proliferation. Despite mutational activation of PIK3CA, melanoma cells were more sensitive to the biochemical and antiproliferative effects of broader spectrum PI3K inhibitors than to an α‐selective PI3K inhibitor. Combined pharmacological inhibition of MEK1/2 and PI3K signaling elicited more potent antiproliferative effects and greater inhibition of the cell division cycle compared to single‐agent inhibition of either pathway alone. Analysis of signaling downstream of MEK1/2 or PI3K revealed that these pathways cooperate to regulate cell proliferation through mTORC1‐mediated effects on ribosomal protein S6 and 4E‐BP1 phosphorylation in an AKT‐dependent manner. Although PI3K inhibition resulted in cytostatic effects on xenografted NRAS^Q61H/PIK3CA^H1047R melanoma, combined inhibition of MEK1/2 plus PI3K elicited significant melanoma regression. This study provides insights as to how mutationally activated PIK3CA acts in concert with MEK1/2 signaling to cooperatively regulate mTORC1/2 to sustain PIK3CA‐mutated melanoma proliferation.     

Retracted: Curcumin ameliorates atherosclerosis through upregulation of miR-126

The potential usage of curcumin in diverse human diseases has been widely studied, including arteriosclerosis (AS). This study focused on investigating the relationship between curcumin and AS-associated microRNA, which may provide a better understanding of curcumin in a different mechanism. Human microvascular endothelial HMEC-1 cells were treated by curcumin alone or oxidized low-density lipoprotein (ox-LDL) plus curcumin, after which the following parameters were analyzed: cell viability, migration, and the expression of AS-associated factors. The regulatory effects of curcumin on miR-126 and signaling pathways involved in AS were then studied. Further, an animal model of AS was stimulated by feeding rabbits with 1% cholesterol diet. The effects of curcumin on the animal model were explored. We found that curcumin treatment significantly reduced HMEC-1 cells viability, migration, and the protein levels of MMP-2, MMP-9, and vascular endothelial growth factor (VEGF) in the presence or absence of ox-LDL. Meanwhile, the expression of VEGFR1 and VEGFR2 was repressed by curcumin. miR-126 was upregulated by curcumin. The abovementioned effects of curcumin on HMEC-1 cells were all attenuated when miR-126 was silenced. And also, VEGF was a target gene of miR-126, and curcumin could inhibit the activation of PI3K/AKT JAK2/STAT5 signaling pathways via miR-126. The effects of curcumin and its regulation on miR-126 and VEGF were confirmed in the animal model of AS. To sum up, curcumin exerted potent anti-AS property possibly via upregulating miR-126 and thereby inhibiting PI3K/AKT and JAK2/STAT5 signaling pathways.     

Ganoderic acid A holds promising cytotoxicity on human glioblastoma mediated by incurring apoptosis and autophagy and inactivating PI3K/AKT signaling pathway

Ganoderic acid A (GA‐A), recognized as a lanostanetriterpene isolated from Ganoderma lucidum, demonstrates an efficient antitumor activity in multiple cancers. To date, it is unclear whether and how GA‐A functions on human glioblastoma (GBM). To unravel the functional significance of GA‐A on human glioblastoma (GBM), the cell‐counting kit‐8 and transwell assays were used to detect proliferation, migration, and invasion of human GBM cell after GA‐A treatment. Then, we utilized the flow cytometry and western blot to further evaluate the effect of GA‐A on GBM cell. Further, activities of autophagy and PI3K/AKT signaling were assessed by Western blot assay. We found that GA‐A significantly inhibited proliferation, migration, and invasion of GBM cell. Additionally, GA‐A markedly triggered cell apoptosis, which incarnated an elevation trend in apoptotic percentage, simultaneously, an increased level of proapoptosis protein (Bax and active caspase‐3) and a decreased level of antiapoptosis protein (Bcl‐2), induced by GA‐A treatment. Meanwhile, levels of two well‐known autophagy markers (beclin 1 and LC3 II) increased while another autophagic substrate (P‐62) was reduced. Moreover, the expressions levels of phosphorylated AKT, mTOR, p‐P70S6K, and cyclin D1 in the PI3K/AKT pathway were significantly reduced, which revealed GA‐A repressed the activation of PI3K/AKT signaling pathway. Collectively, these results indicate that GA‐A may encourage U251 cell growth and invasion/migration inhibition, apoptosis, and autophagy through the inactivation of PI3K/AKT signaling pathway in human GBM. Hence, GA‐A may be a potent antitumorigenic agent for human GBM in future clinical practice.     

过表达MicroRNA-21通过PTEN/PI3K/AKT信号通路调控人退变髓核细胞自噬的研究

目的:探讨过表达mi R-21通过PTEN/PI3K/AKT通路对人退变髓核细胞自噬的影响。方法:构建稳定过表达mi R-21 mimic人退变髓核细胞,转染无意义序列作为mi R-21 mimic control组,采用RT-qPCR检测转染效率;利用MDC荧光染色法观察细胞自噬泡;Western-Blot检测细胞自噬相关蛋白LC3和P62的表达以及PTEN/PI3K/AKT信号通路中关键蛋白PTEN、PI3K及AKT的表达水平。结果:RT-qPCR结果表明mi R-21 mimic转染成功且效率较高,与mi R-21 mimic control组及空白细胞对照组相比,差异显著(P0.05)。荧光显微镜观察MDC染色情况,mi R-21 mimic组的细胞中几乎没有发现自噬体,而mi R-21 mimic control组以及空白对照组细胞中自噬体均较多,与前者相比差异均明显,具有统计学意义(P0.05)。mi R-21 mimic组细胞中LC3-II/LC3-I表达量的比值均显著低于mi R-21 mimic control组及空白对照组细胞(P0.05);而P62在mi R-21 mimic组细胞中表达量显著高于mi R-21 mimic control组及空白细胞对照组,具有统计学意义(P0.05)。mi R-21 mimic组中PTEN蛋白的表达水平较低,与另外两组相比具有统计学意义(P0.05);磷酸化的PI3K(p-PI3K)和AKT(p-Akt)在mi R-21 mimic组中均明显高于mi R-21 mimic control组和空白细胞对照组,差异具有统计学意义(P0.05)。结论:mi R-21可以通过靶向沉默PTEN,促进PI3K和AKT发生磷酸化,进而使PTEN/PI3K/AKT信号通路被激活,最终抑制人椎间盘退变髓核细胞的自噬。     

Regulation of cell growth by autophagy

Cell growth–the primary determinant of cell size–has an intimate relationship with proliferation; cells divide only after they reach a critical size. Despite its developmental and medical significance, little is known about cellular pathways that mediate the growth of cells. Accumulating evidence demonstrates a role for autophagy–a mechanism of eukaryotic cells to digest their own constituents during development or starvation–in cell size control. Increasing autophagic activity by prolonged starvation, rapamycin treatment inhibiting TOR (target of rapamycin) signaling, or genetic intervention, causes cellular atrophy in worms, flies and mammalian cell cultures. In contrast, we have shown that in the nematode Caenorhabditis elegans mutational inactivation of two autophagy genes, unc-51/Atg1 and bec-1/Atg6, confers reduced cell size. We argue that physiological levels of autophagy are required for normal cell size, whereas both insufficient and excessive levels of autophagy lead to retarded cell growth. Furthermore, we discuss data suggesting that the insulin/IGF-1 (insulin-like growth factor receptor-1) and TGFβ (transforming growth factor-beta) signaling systems acting as major growth regulatory pathways converge on autophagy genes to control cell size. Thus, autophagy may act as a central regulatory mechanism of cell growth.

Addendum to: Aladzsity I, Tóth ML, Sigmond T, Szabó E., Bicsák B, Barna J, Reg?s A, Orosz L, Kovács AL, Vellai T. Autophagy genes unc-51 and bec-1 are required for normal cell size in Caenorhabditis elegans. Genetics 2007; 177:655-60, DOI: 10.1534/genetics.107.075762