Development Of Acidic Vesicular Organelles Research Articles

Abstract 4837: Short-chain fatty acids induced autophagy serves as an adaptive strategy for retarding mitochondria-mediated apoptotic cell death

Abstract Short-chain fatty acids (SCFAs) are the major by-products of the bacterial fermentation of undigested dietary fiber in the human large intestine. The role of SCFAs, mostly propionate and butyrate, in colon cancer apoptotic cell death and cell proliferation inhibition has been well studied. Herein we demonstrate that, in addition to apoptotic cell death, propionate and butyrate induce another type of cell death termed “autophagy” in human colon cancer cells. Propionate-treated cells exhibited extensive characteristics of autophagic proteolysis: increased LC3-I to LC3-II conversion and reduced expression of p62/SQSTM1; increased acidic vesicular organelle development as manifested by dramatic increase in punctate GFPLC3, LysoTracker and MDC staining. Propionate-induced autophagy was associated with decreased mTOR activity and enhanced AMP kinase activity, two events known to be linked to autophagy. The elevated AMPKa phosphorylation is believed to have been caused by cellular ATP depletion due to mitochondrial dysfunction involving induction of mitochondrial permeability transition, as evidenced by the loss of mitochondrial membrane potential and overproduction of reactive oxygen species. In this context, mitochondria biogenesis was initiated to recover cellular energy homeostasis. Importantly, when autophagy was prevented at an early stage by 3-methyladenine, or when autophagic degradation was inhibited at a late stage by Chloroquine, the colon cancer cells became sensitized toward propionate induced apoptosis through activation of caspase 7 and its downstream effector caspase-3. Potentiation of cell death was also observed when autophagy was abolished using shRNA against the autophagic gene ATG5. These observations indicate that propionate-triggered autophagy serves as an adaptive strategy for retarding mitochondria-mediated apoptotic cell death. As SCFAs widely exist in the human large intestine, application of an autophagy inhibitor, such as Chloroquine, is expected to enhance the therapeutic efficacy of SCFAs in inducing tumor cell apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4837.

Abstract C8: Short-chain fatty acids induce autophagy through inhibition of mTOR pathway

Abstract Short-chain fatty acid (SCFA), mainly propionate and butyrate are major by-products of the bacterial fermentation of undigested dietary fiber in the human large intestine. Evidence to date implicates propionate/butyrate as inhibitors of colon cancer cell proliferation and inducers of apoptotic cell death. In this study, we demonstrated that propionate/butyrate was able to induce another type of programmed cell death named autophagy in human colon cancer HCT116 cells. Propionate/butyrate-treated cells exhibited extensive characteristics of autophagy, namely the conversion of LC3-I into LC3-II, reduced expression of p62/ SQSTM1; redistribution of GFPLC3 fusion protein from a diffuse pattern towards autophagosomes, which became visible as cytoplasmic puncta; and increased acidic vesicular organelle development as manifested by dramatically increased quantity of strongly stained punctate areas reflected by LysoTracker staining and monodansylcadaverine (MDC) incorporation. Propionate/butyrate-induced autophagy was based on inhibition of its negative regulator-mTOR pathway as evidenced by dephosphorylation of p70S6K and 4E-BP1 and was marked by increased phosphorylation of AMP kinase, two events known to be linked to autophagy. Activation of AMPK was associated with a fluctuation of intracellular ATP, during which the level of ATP transiently increased following propionate/butyrate exposure, reaching maximum at 5 hours, and then declined in slow but gradual manner. The decline of ATP may be due to compromised mitochondria membrane potential and enhanced cellular reactive oxygen species generation. We also showed that this autophagic increase was associated with a series of changes in metabolic pathways, in which energy-demanding program (fatty acids composition and glycogen synthesis inhibition) was downregulated whereas energy-producing program (mitochondria biogenesis) was initiated to cope with the energy crisis. Finally, we showed that increased autophagy and mTOR inhibition by propionate/butyrate are not dependent on p53 activation. Altogether, this study demonstrates autophagy as a novel programmed cell death pathway for propionate/butyrate induced cell death. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C8.

Induction of autophagy by anthrax lethal toxin

Autophagy is an evolutionary conserved intracellular process whereby cells break down long-lived proteins and organelles. Accumulating evidences suggest increasing physiological significance of autophagy in pathogenesis of infectious diseases. Anthrax lethal toxin (LT) exerts its influence on numerous cells and herein, we report a novel effect of LT-induced autophagy on mammalian cells. Several autophagy biochemical markers including LC3-II conversion, increased punctuate distribution of GFP-LC3 and development of acidic vesicular organelles (AVO) were detected in cells treated with LT. Analysis of individual LT component revealed a moderate increase in LC3-II conversion for protective antigen-treated cells, whereas the LC3-II level in lethal factor-treated cells remained unchanged. In addition, our preliminary findings suggest a protective role of autophagy in LT intoxication as autophagy inhibition resulted in accelerated cell death. This study presents a hitherto undescribed effect of LT-induced autophagy on cells and provides the groundwork for future studies on the implication of autophagy in anthrax pathogenesis.

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.

Inhibition of N-(4-hydroxyphenyl)retinamide-induced autophagy at a lower dose enhances cell death in malignant glioma cells

The question whether chemotherapy-induced autophagy is causative to the demise of the cells or a part of the survival mechanism activated during cellular distress is unclear. Others and we have previously demonstrated apoptosis-inducing capacity of N-(4-hydroxyphenyl)retinamide (4-HPR) in malignant glioma cells. We provide evidences of 4-HPR-induced autophagy at a lower concentration (5 microM). Suboptimal dose of 4-HPR treatment of malignant glioma cell lines increased G(2)/M arrest, whereas cell accumulated in S phase at a higher concentration. 4-HPR-induced autophagy was associated with acidic vacuole [acidic vesicular organelle (AVO)] formation and recruitment of microtubule-associated protein light chain 3 (LC3). At a higher concentration of 10 microM of 4-HPR, glioma cells undergoing apoptosis manifested autophagic features indicated by autophagosome formation, AVO development and LC3 localization. Autophagy inhibition at an early stage by 3-methyl adenine inhibited the AVO formation and LC3 localization with an enhancement in cell death. Bafilomycin A1, a specific inhibitor of vacuolar type Hthorn-ATPase also prevented AVO formation without effecting LC-3 localization pattern and also enhanced the extent of 4-HPR-induced cell death. 4-HPR activated c-jun and P38(MAPK) at both 5 and 10 microM concentrations, whereas increased activation of extracellular signal-regulated kinase 1/2 and NF-kappaB was seen only at lower dose. Inhibiting phosphoinositide 3-kinase and mitogen-activated protein kinases pathways modulated 4-HPR-induced cell death. This is the first report that provides evidences that besides apoptosis induction 4-HPR can also induce autophagy. These results indicate that 4-HPR-induced autophagy in glioma cell may provide survival advantage and inhibition of autophagy may enhance the cytotoxicity to 4-HPR.

Autophagic Cell Death of Malignant Glioma Cells Induced by a Conditionally Replicating Adenovirus

Conditionally replicating adenoviruses (CRAds) can be engineered to replicate selectively in cancer cells and cause cancer-specific cell lysis; thus they are considered a promising cancer therapy. To elucidate the mechanisms by which CRAds induce cancer-specific cell death, we infected normal human fibroblasts (MRC5, telomerase negative), human malignant glioma (U373-MG and U87-MG), human cervical cancer (HeLa), and human prostate cancer (PC3) cells (all telomerase positive) with CRAds regulated by the human telomerase reverse transcriptase promoter (hTERT-Ad) or control nonreplicating adenoviruses (Ad-GFP). Nonapoptotic autophagy was assessed in Ad-GFP- and hTERT-Ad-infected cells by examining cell morphology, the development of acidic vesicular organelles, and the conversion of microtubule-associated protein 1 light chain 3 from the cytoplasmic form to the autophagosome membrane form; signaling via mammalian target of rapamycin (mTOR), an autophagy-associated molecule, was monitored by western blot analysis. We also compared the growth of subcutaneous gliomas in nude mice that were treated by intratumoral injection with Ad-GFP or hTERT-Ad. Survival of athymic mice carrying intracranial gliomas treated by intratumoral injection with Ad-GFP or hTERT-Ad was compared by using the Kaplan-Meier method and the Cox-Mantel log-rank analysis. All statistical tests were two-sided. hTERT-Ad induced tumor-specific autophagic cell death in tumor cells and in subcutaneous gliomas. hTERT-Ad-induced autophagy was associated with hTERT-Ad infection kinetics. The mTOR signaling pathway was suppressed in tumor cells and in subcutaneous gliomas treated with hTERT-Ad compared with GFP-Ad or no treatment as shown by reduced phosphorylation of mTOR's downstream target p70S6 kinase (p70S6K). hTERT-Ad treatment of mice (n = 7) slowed growth of subcutaneous gliomas (mean tumor volume = 39 mm3, 95% confidence interval [CI] = 23 to 54 mm3) compared with GFP-Ad treatment (n = 7) (mean tumor volume = 200 mm3, 95% CI = 149 to 251 mm3) at day 7 (volume difference = 161 mm3, 95% CI = 126 to 197 mm3; P < .001). Mice carrying intracranial tumors that were treated with three intratumoral injections of hTERT-Ad survived longer (53 days) than after treatment with GFP-Ad (29 days) (seven mice per group, difference = 24 days, 95% CI = 20 to 28 days; P < .001). hTERT-Ad may kill telomerase-positive cancer cells by inducing autophagic cell death.

Inhibition of platelet-derived growth factor signalling induces autophagy in malignant glioma cells.

Malignant gliomas highly coexpress platelet-derived growth factor (PDGF) and its receptor, suggesting the presence of an autocrine loop. Therefore, disruption of PDGF ligand/receptor complex represents a promising strategy for the treatment of malignant gliomas. However, the mechanisms of the antitumour effect exerted by the inhibition of PDGF-mediated cell growth remain unclear. In the present study, using anti-PDGF neutralising antibody, we investigated the effect of the inhibition of PDGF signalling on malignant glioma U87-MG, D54, and T98G cells with high levels of PDGF-A and -B. As a control, normal fibroblast MRC5 cells expressing low levels of PDGF-A and -B were used. Treatment with anti-PDGF neutralising antibody did not affect the expressions of PDGF-A, PDGF-B, and Akt, but suppressed the level of phosphorylated Akt in tumour cells, indicating the inhibition of PDGF signalling. The cell viability of all malignant glioma cells tested in this study was significantly inhibited in a time-dependent manner following the treatment compared to that of fibroblast cells (P<0.02 to <0.05). The antitumour effect of anti-PDGF antibody was suppressed by the activation of Akt and enhanced by the downregulation of Akt. Interestingly, the inhibition of PDGF signalling induced the development of acidic vesicular organelles and the autophagosome membrane association of the microtubule-associated protein light chain 3, which are characteristic of autophagy, in malignant glioma cells, while apoptotic cell death was not observed. Together these findings imply a new concept of autophagy for PDGF autocrine inhibition in malignant gliomas.