Autophagy plays a pro-survival role against methamphetamine-induced apoptosis in H9C2 cells.

Methamphetamine (METH) is a commonly abused psychostimulant that can induce severe neurotoxicity. Cardiovascular injury caused by METH has recently gained increasing attention; however, the underlying mechanisms remain unclear. As autophagy has been shown to be associated with cell injury, the association between autophagy and METH-mediated cell apoptosis was investigated in the present study. METH treatment significantly increased the expression of two key autophagy proteins, i.e., Beclin-1 and LC3-II, in the cardiomyocyte cell line H9C2. Furthermore, according to western blot and flow cytometry analyses, METH contributed to cell injury and markedly enhanced cleaved-caspase 3 and PARP expression. In addition, the corresponding AKT-mTOR survival pathway axis was appeared deactivated. The autophagic activity was closely associated with METH-mediated cell injury because rapamycin, which is an autophagy inducer, markedly attenuated METH-induced cell injury, while 3-Methyladenine (3-MA), which is an autophagy inhibitor, and bafilomycinA1 (Baf-A1), which is a blocker of autophagosome-lysosome fusion, markedly exacerbated METH-induced cell injury. Notably, defective autophagosome-lysosome fusion might be partially involved in the METH-induced enhancement of LC3-II expression and cell injury. However, the underlying mechanisms require further investigation.

In vitro antitumor effect of a lignan isolated from Combretum fruticosum, trachelogenin, in HCT-116 human colon cancer cells.

The use of natural products in therapeutics has been growing over the years. Lignans are compounds with large pharmaceutical use, which has aroused interest in the search for new drugs to treat diseases. The present study evaluated the cytotoxicity of (-)-trachelogenin, a dibenzylbutyrolactone type lignan isolated from Combretum fruticosum, against several tumor and non-tumor cell lines using the MTT assay and its possible mechanism of action. (-)-Trachelogenin showed IC50 values ranging of 0.8-32.4µM in SF-295 and HL-60 cell lines, respectively and IC50 values >64µM in non-tumor cell lines. (-)-trachelogenin persistently induced autophagic cell death, with cytoplasmic vacuolization and formation of autophagosomes mediated by increasing LC3 activation and altering the expression levels of Beclin-1.

The induction of apoptosis and autophagy in human hepatoma SMMC-7721 cells by combined treatment with vitamin C and polysaccharides extracted from Grifola frondosa.

Polysaccharides extracted from the mushroom Grifola frondosa (GFP) are a potential anticancer agent. The objective of this study was to investigate the effect of GFP and vitamin C (VC) alone and in combination on the viability of human hepatocarcinoma SMMC-7721 and HepG2 cells. Studies designed to detect cell apoptosis and autophagy were also conducted to investigate the mechanism. Results from the cell viability assay indicated that a combination of GFP (0.2 or 0.25 mg/mL) and VC (0.3 mmol/L) (GFP/VC) led to 52.73 and 53.93% reduction in cell viability of SMMC-7721 and HepG2 cells separately after 24 h. Flow cytometric analysis indicated that GFP/VC treatment induced cell cycle arrest at the G2/M phase, and apoptosis occurred in approximately 43.62 and 42.46% of the SMMC-7721 and HepG2 cells separately. Moreover, results of Hoechst33258 and monodansylcadaverine staining, and transmission electron microscopy, showed that GFP/VC induced apoptosis and autophagy in SMMC-7721 and HepG2 cells. Western blot analysis showed changes in the expression of apoptosis-related proteins [upregulation of BAX and caspase-3, downregulation of Bcl-2, and activation of poly-(ADP-ribose)-polymerase] and autophagy protein markers (upregulation of beclin-1 and microtubule-associated protein 1A/1B light chain-3). We also demonstrated that the expression of both Akt and p-Akt was enhanced, suggesting the PI3K/Akt/mTOR pathway might not be involved in this process. Our study shows that the combined application of GFP and VC induced cell apoptosis and autophagy in vitro, and might have antitumor activity in vivo.

Verteporfin inhibits growth of human glioma in vitro without light activation.

Verteporfin (VP), a light-activated drug used in photodynamic therapy for the treatment of choroidal neovascular membranes, has also been shown to be an effective inhibitor of malignant cells. Recently, studies have demonstrated that, even without photo-activation, VP may still inhibit certain tumor cell lines, including ovarian cancer, hepatocarcinoma and retinoblastoma, through the inhibition of the YAP-TEAD complex. In this study, we examined the effects of VP without light activation on human glioma cell lines (LN229 and SNB19). Through western blot analysis, we identified that human glioma cells that were exposed to VP without light activation demonstrated a downregulation of YAP-TEAD-associated downstream signaling molecules, including c-myc, axl, CTGF, cyr61 and survivin and upregulation of the tumor growth inhibitor molecule p38 MAPK. In addition, we observed that expression of VEGFA and the pluripotent marker Oct-4 were also decreased. Verteporfin did not alter the Akt survival pathway or the mTor pathway but there was a modest increase in LC3-IIB, a marker of autophagosome biogenesis. This study suggests that verteporfin should be further explored as an adjuvant therapy for the treatment of glioblastoma.

p53 Mediates Colistin-Induced Autophagy and Apoptosis in PC-12 Cells.

The mechanism of colistin-induced neurotoxicity is still unknown. Our recent study (L. Zhang, Y. H. Zhao, W. J. Ding, G. Z. Jiang, Z. Y. Lu, L. Li, J. L. Wang, J. Li, and J. C. Li, Antimicrob Agents Chemother 59:2189-2197, 2015, http://dx.doi.org/10.1128/AAC.04092-14; H. Jiang, J. C. Li, T. Zhou, C. H. Wang, H. Zhang, and H. Wang, Int J Mol Med 33:1298-1304, 2014, http://dx.doi.org/10.3892/ijmm.2014.1684) indicates that colistin induces autophagy and apoptosis in rat adrenal medulla PC-12 cells, and there is interplay between both cellular events. As an important cellular stress sensor, phosphoprotein p53 can trigger cell cycle arrest and apoptosis and regulate autophagy. The aim of the present study was to investigate the involvement of the p53 pathway in colistin-induced neurotoxicity in PC-12 cells. Specifically, cells were treated with colistin (125 µg/ml) in the absence and presence of a p53 inhibitor, pifithrin-α (PFT-α; 20 nM), for 12 h and 24 h, and the typical hallmarks of autophagy and apoptosis were examined by fluorescence/immunofluorescence microscopy and electron microscopy, real-time PCR, and Western blotting. The results indicate that colistin had a stimulatory effect on the expression levels of the target genes and proteins involved in autophagy and apoptosis, including LC3-II/I, p53, DRAM (damage-regulated autophagy modulator), PUMA (p53 upregulated modulator of apoptosis), Bax, p-AMPK (activated form of AMP-activated protein kinase), and caspase-3. In contrast, colistin appeared to have an inhibitory effect on the expression of p-mTOR (activated form of mammalian target of rapamycin), which is another target protein in autophagy. Importantly, analysis of the levels of p53 in the cells treated with colistin revealed an increase in nuclear p53 at 12 h and cytoplasmic p53 at 24 h. Pretreatment of colistin-treated cells with PFT-α inhibited autophagy and promoted colistin-induced apoptosis. This is the first study to demonstrate that colistin-induced autophagy and apoptosis are associated with the p53-mediated pathway.

The renoprotective role of autophagy activation in proximal tubular epithelial cells in diabetic nephropathy.

With intensive investigations recently, autophagy is hoped to be a potential therapeutic target to prevent or alleviate diabetic nephropathy (DN). Our previous study revealed that lipotoxicity participated in epithelial-to-mesenchymal transition (EMT) of proximal tubular epithelial cells (PTECs) under diabetic conditions. Based on evidences that autophagy and lipid metabolism are closely related, we investigated autophagy under diabetic conditions and how it contributed in the lipotoxicity and EMT. In high-glucose-cultured PTECs, we found that Beclin1 and LC3-II were elevated, while p62 was decreased. These results indicate that autophagy activity was elevated under diabetic conditions. Autophagy deficiency induced by autophagy inhibitors, chloroquine diphosphate (CQ) and 3-Methyladenine (3-MA), and by Atg5 siRNA transfection exacerbated lipid accumulation and EMT. This supports that the elevated autophagy activity acts as a renoprotective response under diabetic conditions. Treatment of rapamycin, which is a mammalian target of rapamycin (mTOR) receptor-specific inhibitor and a known autophagy activator, attenuated high-glucose-induced lipid accumulation and EMT. The Atg5 silence counteracted the protective effect of rapamycin. The present study deepens our understanding of the role of autophagy in DN, suggesting a complex interplay of autophagy, metabolic pathways, lipotoxicity and EMT.

Proteomic analysis of 3-MCPD and 3-MCPD dipalmitate toxicity in rat testis.

Thermal treatment of foodstuff containing fats and salt promotes the formation of 3-chloropropane-1,2-diol (3-MCPD) and its fatty acid esters. 3-MCPD-exposed rats develop testicular lesions and Leydig cell tumors. 3-MCPD and 3-MCPD ester toxicity is thought to be caused by 3-MCPD and its metabolites, since 3-MCPD esters are hydrolyzed in the gut. Inhibition of glycolysis is one of the few known molecular mechanisms of 3-MCPD toxicity. To obtain deeper insight into this process, a comparative proteomic approach was chosen, based on a 28-days repeated-dose feeding study with male Wistar rats. Animals received equimolar doses of 3-MCPD or 3-MCPD dipalmitate. A lower dose of 3-MCPD dipalmitate was also administered. Absence of histopathological changes supported an analysis of early cellular disturbance. Testes were analyzed by two-dimensional gel electrophoresis followed by mass-spectrometric protein identification. Data provide a comprehensive overview of proteomic changes induced by 3-MCPD and 3-MCPD dipalmitate in rat testis in an early phase of organ impairment. Results are compatible with known 3-MCPD effects on reproductive function, substantially extend our knowledge about cellular responses to 3-MCPD and support the hypothesis that toxicity of 3-MCPD and 3-MCPD esters is mediated via common effectors. DJ-1 was identified as a candidate marker for 3-MCPD exposure.

Bergamot polyphenol fraction prevents nonalcoholic fatty liver disease via stimulation of lipophagy in cafeteria diet-induced rat model of metabolic syndrome.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in industrialized countries. Defective autophagy of lipid droplets (LDs) in hepatocytes, also known as lipophagy, has recently been identified as a possible pathophysiological mechanism of NAFLD. Experimental and epidemiological evidence suggests that dietary polyphenols may prevent NAFLD. To address this hypothesis and analyze the underlying mechanisms, we supplemented bergamot polyphenol fraction (BPF) to cafeteria (CAF) diet-fed rats, a good model for pediatric metabolic syndrome and NAFLD. BPF treatment (50 mg/kg/day supplemented with drinking water, 3 months) potently counteracted the pathogenic increase of serum triglycerides and had moderate effects on blood glucose and obesity in this animal model. Importantly, BPF strongly reduced hepatic steatosis as documented by a significant decrease in total lipid content (-41.3% ± 12% S.E.M.), ultrasound examination and histological analysis of liver sections. The morphometric analysis of oil-red stained sections confirmed a dramatic reduction in LDs parameters such as total LD area (48.5% ± 15% S.E.M.) in hepatocytes from CAF+BPF rats. BPF-treated livers showed increased levels of LC3 and Beclin 1 and reduction of SQSTM1/p62, suggesting autophagy stimulation. Consistent with BPF stimulation of lipophagy, higher levels of LC3II were found in the LD subcellular fractions of BPF-expose livers. This study demonstrates that the liver and its lipid metabolism are the main targets of bergamot flavonoids, supporting the concept that supplementation of BPF is an effective strategy to prevent NAFLD.

Pregnenolone activates CLIP-170 to promote microtubule growth and cell migration.

Pregnenolone (P5) is a neurosteroid that improves memory and neurological recovery. It is also required for zebrafish embryonic development. However, its mode of action is unclear. Here we show that P5 promotes cell migration and microtubule polymerization by binding a microtubule plus end-tracking protein, cytoplasmic linker protein 1 (CLIP-170). We captured CLIP-170 from zebrafish embryonic extract using a P5 photoaffinity probe conjugated to diaminobenzophenone. P5 interacted with CLIP-170 at its coiled-coil domain and changed it into an extended conformation. This increased CLIP-170 interaction with microtubules, dynactin subunit p150(Glued) and LIS1; it also promoted CLIP-170-dependent microtubule polymerization. CLIP-170 was essential for P5 to promote microtubule abundance and zebrafish epiboly cell migration during embryogenesis, and overexpression of the P5-binding region of CLIP-170 delayed this migration. P5 also sustained migration directionality of cultured mammalian cells. Our results show that P5 activates CLIP-170 to promote microtubule polymerization and cell migration.

Long term induction by pterostilbene results in autophagy and cellular differentiation in MCF-7 cells via ROS dependent pathway.

This study shows the effect of pterostilbene on intracellular neutral lipid accumulation in MCF-7 breast cancer cells leading to growth arrest and autophagy. On exposing the breast cancer cells with 30 µM pterostilbene for 72 h there was almost 2-folds increase in neutral lipids and triglycerides. Also the phytochemical caused a 4-folds increase in the expression of adipogenic differentiation marker c/EBPα. Further, pterostilbene inhibited 3ß-hydroxylsterol-Δ(7)-reductase, the enzyme which catalyzes the last step conversion of 7-dehydrocholesterol to cholesterol, and thereby causes the intracellular accumulation of the former sterol. These results were associated with over-expression of oxysterol binding protein homologue and liver X receptor (LXR) by ~7-folds. Pterostilbene also caused a simultaneous increase in the expression autophagic marker proteins Beclin 1 and LC3 II (microtubule-associated protein 1 light chain 3) by approximately 6-folds, which leads to an alternative pathway of autophagy. These effects were observed in association with the loss of mitotic and metastatic potential of MCF-7 cells which was abolished in the presence of catalase (ROS scavenger) or 3MA (autophagic inhibitor). Thus the present data shows that the long term exposure to pterostilbene causes growth arrest in MCF-7 cells which may be due to differentiation of the mammary carcinoma cells into normal epithelial cell like morphology and activation of autophagy.

Cell death induced by dexamethasone in lymphoid leukemia is mediated through initiation of autophagy.

Glucocorticoids are fundamental drugs used in the treatment of lymphoid malignancies with apoptotic cell death as the hitherto proposed mechanism of action. Recent studies, however, showed that an alternative mode of cell death, autophagy, is involved in the response to anticancer drugs. The specific role of autophagy and its relationship to apoptosis remains, nevertheless, controversial: it can either lead to cell survival or can function in cell death. We show that dexamethasone induced autophagy upstream of apoptosis in acute lymphoblastic leukemia cells. Inhibition of autophagy by siRNA-mediated repression of Beclin 1 expression inhibited apoptosis showing an important role of autophagy in dexamethasone-induced cell death. Dexamethasone treatment caused an upregulation of promyelocytic leukemia protein, PML, its complex formation with protein kinase B or Akt and a PML-dependent Akt dephosphorylation. Initiation of autophagy and the onset of apoptosis were both dependent on these events. PML knockout thymocytes were resistant to dexamethasone-induced death and upregulation of PML correlated with the ability of dexamethasone to kill primary leukemic cells. Our data reveal key mechanisms of dexamethasone-induced cell death that may inform the development of improved treatment protocols for lymphoid malignancies.

Microtubule-associated protein 2 (MAP2) is a neurosteroid receptor.

The neurosteroid pregnenolone (PREG) and its chemically synthesized analog 3beta-methoxypregnenolone (MePREG) bind to microtubule-associated protein 2 (MAP2) and stimulate the polymerization of microtubules. PREG, MePREG, and progesterone (PROG; the physiological immediate metabolite of PREG) significantly enhance neurite outgrowth of nerve growth factor-pretreated PC12 cells. However, PROG, although it binds to MAP2, does not increase the immunostaining of MAP2, contrary to PREG and MePREG. Nocodazole, a microtubule-disrupting agent, induces a major retraction of neurites in control cultures, but pretreatment with PREG/MePREG is protective. Decreasing MAP2 expression by RNA interference does not modify PROG action, but it prevents the stimulatory effects of PREG and MePREG on neurite extension, showing that MAP2 is their specific receptor.