Autophagic Effects Research Articles

Impacts of CAD/CAM Metallic Materials on Trace Metals and Biocompatibilities: An in vivo Study in Beagle Dogs

CAD/CAM (computer-aided design and computer-aided manufacturing) technology has been widely applied in clinical dentistry, but the material safety remains a concern. To investigate the impacts of CAD/CAM metallic materials on trace metals and biocompatibilities, selective laser melted (SLM) cobalt-chromium (Co-Cr) alloys and computer numeric controlled milled (CNC milled) commercially pure titanium (CP-Ti) were placed on the maxilla of beagle dogs for 6 months. The trace metals in the oral mucosa, blood, liver, kidney, and hair were then determined by inductively coupled plasma mass spectrometry (ICP-MS). The histopathologic changes and biocompatibilities of tissues were examined by hematoxylin and eosin (H&E) staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labeling (TUNEL) method, immunohistochemistry (IHC) assay, Western blot analysis, and liver and kidney function tests. Our results showed that trace metals released from these two CAD/CAM metallic materials accumulated transiently in the oral mucosa and whole blood. The trace metals released from SLM Co-Cr alloys could also transiently accumulate in the plasm and hair. In addition, these two CAD/CAM metallic materials both induced apoptosis and histopathologic changes in the oral mucosa, with SLM Co-Cr alloys inducing a higher level of apoptosis. In contrast, both materials did not exert autophagic effects on the oral mucosa or affect the trace metals, functions, or biocompatibilities of the liver and kidney. Therefore, this study suggests that CAD/CAM metallic materials should be selected carefully, especially in patients with metal- and apoptosis-related diseases, and CNC-milled CP-Ti can be recommended to patients on account of its better biocompatibility and safety.

Dual Akt and Bcl-2 inhibition induces cell-type specific modulation of apoptotic and autophagic signaling in castration resistant prostate cancer cell lines.

Cancer cell survival depends on the cross-regulation between apoptosis and autophagy which share common signaling pathways including PI3K/Akt/mTOR and Bcl-2. The aim of this study was to elucidate the modulation patterns between apoptosis and autophagy following dual inhibition by Akt inhibitor erufosine and Bcl-2 inhibitor ABT-737 in castration-resistant prostate cancer (CRPC) cell lines, PC-3 (Bax+) and DU-145 (Bax-). Cell cycle progression, apoptotic and autophagic signaling were examined by flow cytometry, multi-caspase assay, Hoechst staining, acridine orange staining of acidic vesicular organelles (AVOs), qRT-PCR and Western Blot. Dual inhibition increased G2/M arrest in PC-3 and DU-145, but not in the healthy prostate epithelium cells, PNT-1A. Only in PC-3, dual inhibition induced synergistic apoptotic and additive autophagic effects. In DU-145 and PNT-1A cells, ABT-737 did not display any remarkable effect on multicaspase activity and erufosine and ABT-737, neither alone nor in combination induced AVOs. By dual inhibition, AKT, BCL-2 and NF-κB gene expressions were downregulated in PC-3, both ATG-5 and BECLIN-1 gene expressions were upregulated in DU-145 but Beclin-1 protein expression was substantially reduced in both CRPC cells. Dual inhibition-induced synergistic multicaspase activation in PC-3 degrades and disrupts autophagic activity of Beclin-1, enhancing caspase-dependent apoptosis. However, in DU-145, following dual inhibition, rate of multicaspase induction and apoptosis are lower but autophagy is completely abolished despite markedly increased BECLIN-1 gene expression. In conclusion, antineoplastic drug combinations may display cell-type specific modulation of apoptotic and autophagic signaling and lack of protective autophagy may not necessarily indicate increased chemotherapeutic sensitivity in heterogenous tumor subpopulations.

Cx32 inhibits the autophagic effect of Nur77 in SH-SY5Y cells and rat brain with ischemic stroke.

The pathogenesis of cerebral ischemia-reperfusion (I/R) is complex. Cx32 expression has been reported to be up-regulated in ischemic lesions of aged human brain. Nevertheless, the function of Cx32 during cerebral I/R is poorly understood. Autophagy is of vital importance in the pathogenesis of cerebral I/R. In the current study, we found that oxygen-glucose deprivation/reoxygenation (OGD/R) or I/R insult significantly induced the up-regulation of Cx32 and activation of autophagy. Inhibition of Cx32 alleviated OGD/R or I/R injury, and further activated autophagy. In addition, Nur77 expression was found to be up-regulated after OGD/R or I/R. After inhibiting Cx32, the expression of Nur77 was further increased and Nur77 was translocated from nucleus to mitochondrial. Inhibition of Cx32 also activated mitophagy by promoting autophagosome formation and up-regulating the expression of mitochondrial autophagy marker molecules. Of note, in the siNur77-transfected cells, the number of dysfunctional mitochondrial was increased, and mitophagy was suppressed, which aggravated OGD/R-induced neuronal injury. In conclusion, Cx32 might act as a regulatory factor of Nur77 controlling neuronal autophagy in the brains. Understanding the mechanism of this regulatory pathway will provide new insight into the role Cx32 and Nur77 in cerebral ischemia, offering new opportunities for therapeutics.

Autophagy Promotes the Survival of Adipose Mesenchymal Stem/Stromal Cells and Enhances Their Therapeutic Effects in Cisplatin-Induced Liver Injury via Modulating TGF-β1/Smad and PI3K/AKT Signaling Pathways.

Autophagy is a key metabolic process where cells can recycle its proteins and organelles to regenerate its own cellular building blocks. Chemotherapy is indispensable for cancer treatment but associated with various side-effects, including organ damage. Stem cell-based therapy is a promising approach for reducing chemotherapeutic side effects, however, one of its main culprits is the poor survival of transplanted stem cells in damaged tissues. Here, we aimed to test the effects of activating autophagy in adipose-derived mesenchymal stem/stromal cells (ADSCs) on the survival of ADSCs, and their therapeutic value in cisplatin-induced liver injury model. Autophagy was activated in ADSCs by rapamycin (50 nM/L) for two hours before transplantation and were compared to non-preconditioned ADSCs. Rapamycin preconditioning resulted in activated autophagy and improved survival of ADSCs achieved by increased autophagosomes, upregulated autophagy-specific LC3-II gene, decreased protein degradation/ubiquitination by downregulated p62 gene, downregulated mTOR gene, and finally, upregulated antiapoptotic BCL-2 gene. In addition, autophagic ADSCs transplantation in the cisplatin liver injury model, liver biochemical parameters (AST, ALT and albumin), lipid peroxidation (MDA), antioxidant profile (SOD and GPX) and histopathological picture were improved, approaching near-normal conditions. These promising autophagic ADSCs effects were achieved by modulation of components in TGF-β1/Smad and PI3K-AKT signaling pathways, besides reducing NF-κB gene expression (marker for inflammation), reducing TGF-β1 levels (marker for fibrosis) and increasing SDF-1 levels (liver regeneration marker) in liver. Therefore, current results highlight the importance of autophagy in augmenting the therapeutic potential of stem cell therapy in alleviating cisplatin-associated liver damage and opens the path for improved cell-based therapies, in general, and with chemotherapeutics, in particular.

INVESTIGATION OF THE POSSIBLE EFFECT OF S-ALLYL-L-CYSTEINE ON APOPTOSIS AND AUTOPHAGY IN HUMAN LEUKEMIA CELL LINE

OBJECTIVE: S-Allyl-L-cysteine (SAC) is a biological active organosulfur component of garlic and has various pharmacological effects. SAC has displayed anti-cancer activity but the mechanism is unresolved. This study has focused on investigating the possible apoptotic and autophagic effects of SAC on two human leukemia cell lines: acute promyelocytic leukemia (HL-60) and chronic myeloid leukemia (K562).MATERIAL AND METHODS: Cell cytotoxicity was evaluated via MTT test. Bax, Bcl-2, caspase 3, mTOR, AKT, and PI3K gene expression amounts were identified via Real time quantitative reverse transcription polymerase chain reaction (qRT-PCR). HL-60 and K562 cells were incubated with SAC at three diverse doses (5 mM, 10 mM, and 20 mM) (3,75 mM, 7,5 mM, and 15 mM), respectively.RESULTS: SAC caused a cytotoxic effect on HL-60 and K562 cells with IC50 values of approximately 11.525 mM and 10.025 mM, respectively. In HL-60 cells, an increase in Bax expression levels was detected at doses of 5 mM and 10 mM SAC (p=0.027, p=0.000). Treatment with 10 mM SAC increased the expression level of caspase 3 in HL-60 cells as compared with the control and 5 mM SAC treated cells (p=0.000, p=0.020). In K562 cells, SAC induced a significant decrease in mTOR, AKT, and PI3K expression levels in at all doses (p=0.000, p=0.000, p=0.000).CONCLUSIONS: In conclusion, our data indicates that SAC induces autophagy in K562 cells by downregulating the PI3K/AKT/mTOR signaling pathway. Furthermore, increased Bax and caspase 3 gene expression levels suggest that SAC may be an effective active ingredient with which to induce apoptosis in HL-60 cells.

PPM-18, an Analog of Vitamin K, Induces Autophagy and Apoptosis in Bladder Cancer Cells Through ROS and AMPK Signaling Pathways.

PPM-18, identified as a novel analog of vitamin K, has been reported to play a critical role in the suppression of seizures. However, the concerns that whether PPM-18, like vitamin K, exerts anticancer activity remain to be further investigated. Here, we found that PPM-18 remarkably suppressed the proliferation and induced apoptosis in bladder cancer cells. Furthermore, a significant autophagic effect of PPM-18 on bladder cancer cells was also demonstrated, which profoundly promoted apoptotic cell death. Mechanistically, PPM-18 activated AMP-activated protein kinase (AMPK), whereas it repressed PI3K/AKT and mTORC1 pathways in bladder cancer cells. Inhibition of AMPK markedly relieved PPM-18–induced autophagy and apoptosis, indicating that PPM-18 is able to induce autophagy and apoptosis in bladder cancer cells via AMPK activation. Moreover, reactive oxygen species (ROS) were notably accumulated in PPM-18–treated bladder cancer cells, and treatment with ROS scavengers not only eliminated ROS production but also abrogated AMPK activation, which eventually rescued bladder cancer cells from PPM-18–triggered autophagy and apoptotic cell death. In bladder cancer xenografts, the anticancer activities of PPM-18, including suppressing the growth of tumors and inducing autophagy and apoptosis in tumor cells, were also established. Collectively, this study was the first to demonstrate the anticancer effect of PPM-18 on bladder cancer cells in vitro and in vivo through eliciting autophagy and apoptosis via ROS and AMPK pathways, which might provide new insights into the potential utilization of PPM-18 for future bladder cancer treatment.

Abstract LB184: Induction of ulk1 regulated autophagy by a novel antipsychotic drug leads to apoptosis in pancreatic cancer cells

Abstract Pancreatic cancer patients have limited treatment options in spite of several advanced treatment strategies. Pancreatic tumors exhibit high basal autophagy compared to other cancers. Several studies including from our lab reported that enhanced autophagy leads to apoptosis in cancer cells. In this study, we have demonstrated that pimavanserin (PVT) suppresses pancreatic tumor growth by inducing autophagy-mediated apoptosis. Our results indicated that PVT induced 1.8 - 5 fold autophagy in pancreatic ductal adenocarcinoma (PDAC) cells, as evaluated by increased acridine orange staining. Furthermore, accumulation of double-membrane vesicles (autophagosomes) and formation of autophagolysosomes were observed with PVT treatment. Genomic analysis by R2 &GEPIA database revealed that PDAC patients exhibited reduced levels of autophagy initiator gene, ULK1. PVT induced the expression of autophagy markers ULK1, FIP200, Atg101, Beclin-1, Atg5, and LC3A/B in several pancreatic cancer cells. In addition, phosphorylation of ULK1 atSer757 was inhibited with PVT treatment. Apoptotic effects of PVT in pancreatic cancer cells were validated by increase in cleavage of caspase3. ULK1 agonist LYN-1604 enhanced the autophagic and apoptotic effects of PVT. On the other hand, autophagy inhibitors chloroquine and bafilomycin blocked the autophagic and apoptotic effects of PVT in pancreatic cancer cells. Our in vivo findings demonstrated that chloroquine abrogated the growth-suppressive effects of PVT by 21% in sub-cutaneously implanted BxPC3 tumor xenografts. Moreover, chloroquine reduced the effects of PVT in inducing the expression of ULK1 and the cleavage of caspase 3 as evaluated by western blotting. Oral administration of PVT suppressed BxPC3 tumor xenografts by 50% in athymic nude mice. In another in vivo experiment, PVT treatment inhibited the growth of orthotopically implanted PANC1 tumors by 77%. Chronic administration of PVT did not exhibit any general signs of toxicity or behavioral side effects in mice. Moreover, long-term administration of PVTdid not altered the clinical chemistry parameters like ALT, AST, total serum protein, calcium, creatinine, BUN, and albumin. Collectively, our results indicate that PVT-mediated pancreatic tumor growth suppression was associated with the induction of autophagy-mediated apoptosis. Since PVT is already available in the clinic with an established safety profile, our results will accelerate its clinical development for pancreatic cancer therapy. Citation Format: Sharavan Ramachandran, Sanjay K. Srivastava. Induction of ulk1 regulated autophagy by a novel antipsychotic drug leads to apoptosis in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB184.

Pimavanserin: A novel autophagic regulator for pancreatic cancer treatment

Pancreatic cancer patients have limited treatment options in spite of several advanced treatment strategies. Pancreatic tumors exhibit high basal autophagy compared to other cancers. Several studies including from our lab reported that enhanced autophagy leads to apoptosis in cancer cells. In this study, we have demonstrated that pimavanserin (PVT) suppresses pancreatic tumor growth by inducing autophagy-mediated apoptosis. Our results indicated that PVT induced 1.8 – 5 fold autophagy in pancreatic ductal adenocarcinoma (PDAC) cells, as evaluated by increased acridine orange staining. Furthermore, accumulation of double membrane vesicles (autophagosomes) and formation autophagolysosomes were observed with PVT treatment. Genomic analysis by R2 & GEPIA database revealed that PDAC patients exhibited reduced levels of autophagy initiator gene, ULK1. PVT induced the expression of autophagy markers ULK1, FIP200, Atg101, Beclin-1, Atg5, and LC3A/B in several pancreatic cancer cells. In addition, phosphorylation of ULK1 at Ser757 was inhibited with PVT treatment. Apoptotic effects of PVT in pancreatic cancer cells was validated by increase in cleavage of caspase3. ULK1 agonist LYN-1604 enhanced the autophagic and apoptotic effects of PVT. On the other hand, autophagy inhibitors chloroquine and bafilomycin blocked the autophagic and apoptotic effects of PVT in pancreatic cancer cells. Our in vivo findings demonstrated that chloroquine abrogated the growth suppressive effects of PVT by 21% in sub-cutaneously implanted BxPC3 tumor xenografts. Moreover, chloroquine reduced the effects of PVT in inducing the expression of ULK1 and the cleavage of caspase 3 as evaluated by western blotting. Oral administration of PVT suppressed BxPC3 tumor xenografts by 50% in athymic nude mice. In another in vivo experiment, PVT treatment inhibited the growth of orthotopicaly implanted PANC1 tumors by 77%. Chronic administration of PVT did not exhibit any general signs of toxicity or behavioral side effects in mice. Moreover, long-term administration of PVT did not altered the clinical chemistry parameters like ALT, AST, total serum protein, calcium, creatinine, BUN and albumin. Collectively, our results indicate that PVT mediated pancreatic tumor growth suppression was associated with induction of autophagy mediated apoptosis. Since, PVT is already available in clinic with an established safety profile, our results will accelerate its clinical development for pancreatic cancer therapy.

NGFR Increases the Chemosensitivity of Colorectal Cancer Cells by Enhancing the Apoptotic and Autophagic Effects of 5-fluorouracil via the Activation of S100A9.

Colorectal cancer (CRC) is currently the third leading cause of cancer-related deaths worldwide, and 5-fluorouracil (5-FU)-based chemotherapies serve as important adjuvant therapies before and after surgery for CRC. However, the efficacy of CRC chemotherapy is limited by chemoresistance, and therefore the discovery of novel markers to indicate chemosensitivity is essential. Nerve growth factor receptor (NGFR), a cell surface receptor, is involved in cell death and survival. Our previous study indicated that NGFR acts as a tumor suppressor, and high expression is associated with better outcomes in patients receiving 5-FU-based adjuvant chemotherapy after surgery. The aim of this study was to investigate the effect of NGFR on the chemotherapeutic response in CRC. Chemosensitivity was investigated using DLD1 and HCT8 cells after NGFR transfection. Apoptosis was investigated by flow cytometry. Autophagy was assessed using GFP-LC3B transient transfection. Gene expression was measured using an mRNA microarray. Beclin-1 and Bcl-2 protein expressions were assessed by western blot. NGFR and S100 calcium-binding protein A9 (S100A9) expressions in CRC patients were investigated by immunohistochemistry. The results showed that the half maximal inhibitory concentration of NGFR-transfected cells was lower than that of controls in DLD1 and HCT8 cells after 5-FU treatment, and cell viability was lower than in empty-vector cells. Tumor sizes were also smaller than in empty-vector cells in vivo. The percentages of apoptotic and autophagic cells were higher in NGFR-transfected cells. NGFR elevated the expression of S100A9 after 5-FU treatment. The combination of Bcl-2 and Beclin-1 was significantly suppressed by overexpressed NGFR. Five-year overall and disease-free survival in NGFR+/S100A9+ patients was better than in NGFR−/S100A9− patients. This study’s findings suggest that NGFR may serve as a marker predicting CRC patients’ chemosensitivity.

Total ginsenosides induce autophagic cell death in cervical cancer cells accompanied by downregulation of bone marrow stromal antigen-2.

Ginsenosides are important active components in Panax ginseng. In the present study, total ginsenosides (TGNs) were demonstrated to enhance autophagy by promoting acidic vacuole organelle formation, recruitment of enhanced green fluorescent protein-microtubule-associated protein light chain 3 and expression of autophagy-related factors in cervical cancer cell lines. TGN markedly increased the expression of p62 at the transcriptional level, but decreased p62 protein expression in the presence of actinomycin D. The autophagic regulatory effect was reversible. TGN (≤120 µg/ml) did not affect the proliferation of cervical cancer cells under normal culture conditions, but markedly inhibited the growth of serum-deprived cells. Treatment with an inhibitor of autophagy (3-methyladenine) impaired TGN-induced cell death. This suggested that TGN caused autophagic cell death. In addition, western blot analysis demonstrated that the protein level of bone marrow stromal antigen-2 (BST-2) was downregulated by TGN. Upregulation of BST-2 reduced cell death. The results of the combined actions of various monomeric ginsenosides in TGN provide the molecular basis to develop TGN as a promising candidate for cancer therapy.

The Role of Vitamin K in Humans: Implication in Aging and Age-Associated Diseases

As human life expectancy is rising, the incidence of age-associated diseases will also increase. Scientific evidence has revealed that healthy diets, including good fats, vitamins, minerals, or polyphenolics, could have antioxidant and anti-inflammatory activities, with antiaging effects. Recent studies demonstrated that vitamin K is a vital cofactor in activating several proteins, which act against age-related syndromes. Thus, vitamin K can carboxylate osteocalcin (a protein capable of transporting and fixing calcium in bone), activate matrix Gla protein (an inhibitor of vascular calcification and cardiovascular events) and carboxylate Gas6 protein (involved in brain physiology and a cognitive decline and neurodegenerative disease inhibitor). By improving insulin sensitivity, vitamin K lowers diabetes risk. It also exerts antiproliferative, proapoptotic, autophagic effects and has been associated with a reduced risk of cancer. Recent research shows that protein S, another vitamin K-dependent protein, can prevent the cytokine storm observed in COVID-19 cases. The reduced activation of protein S due to the pneumonia-induced vitamin K depletion was correlated with higher thrombogenicity and possibly fatal outcomes in COVID-19 patients. Our review aimed to present the latest scientific evidence about vitamin K and its role in preventing age-associated diseases and/or improving the effectiveness of medical treatments in mature adults ˃50 years old.

Alcoholic extract of Tarantula cubensis (Theranekron®) induce autophagy on gastric cancer cells

Aim: To evaluate the effects of theranekron in respect of autophagy on gastric cancer that is the fifth leading cancer type worldwide. Metods: In the present study, metastatic AGS and non-metastatic MKN-45 human gastric cell lines were used together with HEK-293 non-cancer cells as controls. Cytotoxic effect of theranekron besides appropriate treatment time was investigated through cell proliferation by using Cell Proliferation assay Kit (MTT) using different concentrations of the drug. The autophagic effect of the drug was determined using the LC3-GFP translocation assay and western blot analysis. All experiments were performed also using the ethanol since Tarantula cubensis spider was processed and diluted in 60% alcohol to generate as a drug. Results: MTT assay results demonstrated that the half maximal inhibitory concentration of theranekron was ~100 μM, its effect was found to be significant at 6 hrs, and theranekron decreased the cell viability in all cell lines without specificity in respect to the increasing concentrations. Additionally, a significantly increased GFP accumulation was detected in the autophagosomes of the cells treated with theranekron compared to non-treated cells, indicating the presence of autophagy. Conclusion: These findings were confirmed by LC3-I to LC3-II conversion with the western blot analysis. The data of ethanol experiments; however, demonstrated that ethanol also induced a cytotoxic effect and autophagic cell death. Our results suggested that theranekron results in cell death and stimulate autophagy process, but it is not specific for cancer cells since it represented similar results on non-cancer control cells. Moreover, the effect of theranekron on cell death might mostly occur through alcohol in which it is extracted.

β-Arrestin inhibition induces autophagy, apoptosis, G0/G1 cell cycle arrest in agonist-activated V2R receptor in breast cancer cells

Non-visual arrestins (β-arrestins) are endocytic proteins that mediate agonist-activated GPCRs internalization and signaling pathways in an independent manner. The involvement of β-arrestins in cancer invasion and metastasis is increasingly reported. So, it is hypothesized that inhibition of β-arrestins may diminish the survival chances of cancer cells. This study aimed to evaluate the in vitro impact of inhibiting β-arrestins on the autophagic and/or apoptotic responsiveness of breast cancer cells. We used Barbadin to selectively inhibit β-Arr/AP2 interaction in AVP-stimulated V2R receptor of triple-negative breast cancer cells (MDA MB-231). Autophagy was assessed by the microtubule-associated protein 1 light chain 3-II (LC3II), apoptosis was measured by Annexin-V/PI staining and cell cycle distribution was investigated based upon the DNA content using flow cytometry. Barbadin reduced cell viability to 69.1% and increased the autophagy marker LC3II and its autophagic effect disappeared in cells transiently starved in Earle's balanced salt solution (EBSS). Also, Barbadin mildly enhanced the expression of P62 mRNA and arrested 63.7% of cells in G0/G1 phase. In parallel, the drug-induced apoptosis in 29.9% of cells (by AV/PI) and 27.8% of cells were trapped in sub-G1 phase. The apoptotic effect of Barbadin was enhanced when autophagy was inhibited by the PI3K inhibitor (Wortmannin). Conclusively, the data demonstrate the dual autophagic and apoptotic effects of β-βArr/AP2 inhibition in triple-negative breast cancer cells. These observations nominate β-Arrs as selective targets in breast cancer treatment.

Beclin-1/LC3-II dependent macroautophagy was uninfluenced in ischemia-challenged vascular endothelial cells

Autophagy has been extensively studied and occurs in many biological settings. However, a question remains as to whether ischemia enhances Beclin-1/LC3-II-dependent macroautophagy in vascular endothelial cells, as has been previously thought. Furthermore, the effect of the level of autophagy on cell or skin flap survival still requires elucidation. We created a lethal ischemia model in human umbilical vascular endothelial cells (HUVECs), performed quantitative proteomics and bioinformatics analyses, and verified the autophagic status and effect both in vitro and in vivo. The significantly upregulated proteins encoded by autophagy-related genes (ATGs) included ATG2A, ATG3, ATG4B, ATG5, ATG7, ATG9A, ATG12, ATG16, and ATG101. The significantly enhanced lysosomal proteins were cathepsin B, cathepsin D, lysosome-associated membrane protein 1 (LAMP1), and LAMP2. However, the differentially expressed proteins excluded Beclin-1, microtubule-associated protein light chain 3 (LC3)-I, and LC3-II. Western blot analyses verified that the protein expression levels of Beclin-1, LC3-I, and LC3-II were neither upregulated nor downregulated in ischemia-challenged HUVECs. The autophagic status was not enhanced by rapamycin in ischemic HUVECs but appeared to be inhibited by chloroquine. Our in vivo study on rats showed that a downregulation in autophagic status jeopardized skin flap survival. In conclusion, Ischemia neither enhanced nor inhibited Beclin-1/LC3-II-dependent canonical macroautophagy both in vitro and in vivo, in contradiction to previous studies. An appropriate autophagic homeostasis can minimize cell or skin flap damage.

Artemisia kruhsiana leaf extract induces autophagic cell death in human prostate cancer cells.

Some species of Artemisia have been reported to induce apoptosis and autophagy, but little is known of the apoptotic and autophagic effects of the stems and leaves of Artemisia kruhsiana Bess. (AkB). This study was conducted to investigate the antioxidant and anti-autophagic effects of the methanol extracts of the stems (EAkBs) and leaves (EAkBl) of AkB on human prostate cancer PC-3 cells. The antioxidant effects of EAkBs and EAkBl were measured using in vitro total flavonoid and total phenolic assays and a free radical scavenging assay. The effects of EAkBl on cell viability, apoptosis, autophagy, intracellular reactive oxygen species (ROS) generation and protein expression levels were also investigated. EAkBl was found to induce apoptosis, autophagy, and intracellular ROS generation in PC-3 cells. In terms of protein levels, EAkBl reduced phospho (p)-protein kinase B (AKT)/AKT, p-mammalian target of rapamycin (mTOR)/mTOR, B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X protein (Bax) ratios, and the activations of beclin 1/β-actin and microtubule-associated protein 1A/1B-light chain 3 (LC3) II/LC3 I ratios in PC-3 cells. The results of this study indicate EAkBl has antioxidant and anticancer effects on prostate cancer cells, and that these effects are associated with suppressions of p-AKT, p-mTOR, Bcl-2, and Bax, and the activations of beclin 1 and LC3. Our results indicate EAkBl has potential as a treatment for prostate cancer.

Polysaccharide from Angelica sinensis attenuates SNP-induced apoptosis in osteoarthritis chondrocytes by inducing autophagy via the ERK1/2 pathway

ObjectiveChondrocyte apoptosis plays a vital role in osteoarthritis (OA) progression. Angelica sinensis polysaccharide (ASP), a traditional Chinese medicine, possesses anti-inflammatory and anti-apoptotic properties in chondrocytes. This study aimed to determine the protective role of ASP on sodium nitroprusside (SNP)-induced chondrocyte apoptosis, and explore the underlying mechanism.MethodHuman primary chondrocytes isolated from the articular cartilage of OA patients were treated with SNP alone or in combination with different doses of ASP. Cell viability and apoptosis were assessed, and apoptosis-related proteins including Bcl-2 and Bax were detected. Autophagy levels were evaluated by light chain 3 (LC3) II immunofluorescence staining, mRFP-GFP-LC3 fluorescence localization, and western blot (LC3II, p62, Beclin-1, Atg5). Meanwhile, activation of the ERK 1/2 pathway was determined by western blot. The autophagy inhibitors, 3-methyladenine (3-MA), chloroquine (CQ), and a specific inhibitor of ERK1/2, SCH772984, were used to confirm the autophagic effect of ASP.ResultsThe results showed that SNP-induced chondrocyte apoptosis was significantly rescued by ASP, whereas ASP alone promoted chondrocyte proliferation. The anti-apoptotic effect of ASP was related to the enhanced autophagy and depended on the activation of the ERK1/2 pathway.ConclusionASP markedly rescued SNP-induced apoptosis by activating ERK1/2-dependent autophagy in chondrocytes, and it made ASP as a potential therapeutic supplementation for OA treatment.

Lactate regulates autophagy through ROS-mediated activation of ERK1/2/m-TOR/p-70S6K pathway in skeletal muscle.

The role of autophagy and lysosomal degradation pathway in the regulation of skeletal muscle metabolism was previously studied. However, underlying molecular mechanisms are poorly understood. L-lactate which is utilized as an energetic substrate by skeletal muscle can also augment genes expression related to metabolism and up-regulate those being responsive to reactive oxygen species (ROS). Since ROS is the most important regulator of autophagy in skeletal muscle, we tested if there is a link between cellular lactate metabolism and autophagy in differentiated C2C12 myotubes and the gastrocnemius muscle of male wistar rats. C2C12 mouse skeletal muscle was exposed to 2, 6, 10, and 20mM lactate and evaluated for lactate autophagic effects. Lactate dose-dependently increased autophagy and augmented ROS generation in differentiated C2C12 myotubes. The autophagic effect of lactate deterred in N-acetylcysteine presence (NAC, a ROS scavenger) indicated lactate regulates autophagy with ROS participation. Lactate-induced up-regulation of extracellular signal-regulated kinase 1/2 (ERK1/2) through ROS was required to regulate the autophagy by lactate. Further analysis about ERK1/2 up- and downstream indicated that lactate regulates autophagy through ROS-mediated the activation of ERK1/2/mTOR/p70S6K pathway in skeletal muscle. The in vitro effects of lactate on autophagy also occurred in the gastrocnemius muscle of male Wistar rats. In conclusion, we provided the lactate-associated regulation evidence of autophagy in skeletal muscle by activating ROS-mediated ERK1/2/mTOR/p70S6K pathway. Since the increase in cellular lactate concentration is a hallmark of energy deficiency, the results provide insight into a skeletal muscle mechanism to fulfill its enhanced energy requirement.

In Vitro Toxicological Assessment of Gadodiamide in Normal Brain SVG P12 Cells.

Magnetic resonance imaging (MRI) is a technique for evaluating patients with primary and metastatic tumors. The contrast agents improve the diagnostic accuracy of MRI. Large quantities of a contrast agent must be administrated into the patient to obtain useful images, which leads to cell injury. Gadolinium has been reported to cause central lobular necrosis of the liver and nephrogenic systemic fibrosis. However, the toxicity caused on brain tissue is uncertain. This study mainly aimed on the in vitro study of high concentration (2 and 5-fold of normal concentration) gadolinium-based contrast agents (GBCAs), gadodiamide (Omniscan®), on normal brain glial SVG P12 cells. MTT assay, DAPI staining, immunofluorescent staining, LysoTracker Red staining, and western blotting analysis were applied on the cells. The viability of gadodiamide (1.3, 2.6, 5.2, 13 and 26 mM)-treated SVG P12 cells was significantly reduced after 24 h of incubation. Gadodiamide caused significant autophagic flux at 2.6, 5.2 and 13.0 mM as seen by acridine orange (AO) staining, LC-3-GFP and LysoTracker Red staining. The expression levels of autophagy-related proteins such as beclin-1, ATG-5, ATG-14 and LC-3 II were up-regulated after 24 h of gadodiamide incubation. Autophagy inhibitors including 3-methyladenine (3-MA), chloroquine (CQ) and bafilomycin A1 (Baf) significantly alleviated the autophagic cell death effect of gadodiamide on normal brain glial SVG P12 cells. Gadodiamide induced significant apoptotic effects at 5.2 mM and 13.0 mM as seen by DAPI staining and the pan-caspase inhibitor significantly alleviated the apoptotic effect. Gadodiamide at 5.2 mM and 13.0 mM inhibited antiapoptotic protein expression levels of Bcl-2 and Bcl-XL, while promoted pro-apoptotic protein expression levels of Bax, BAD, cytochrome c, Apaf-1, cleaved-caspase-9 and cleaved-caspase-3. Normal brain glial SVG P12 cells treated with high concentrations of gadodiamide can undergo autophagy and apoptosis.

Pharmacological Inhibition of O-GlcNAc Transferase Promotes mTOR-Dependent Autophagy in Rat Cortical Neurons.

O-GlcNAc transferase (OGT) is a ubiquitous enzyme that regulates the addition of β-N-acetylglucosamine (O-GlcNAc) to serine and threonine residues of target proteins. Autophagy is a cellular process of self-digestion, in which cytoplasmic resources, such as aggregate proteins, toxic compounds, damaged organelles, mitochondria, and lipid molecules, are degraded and recycled. Here, we examined how three different OGT inhibitors, alloxan, BXZ2, and OSMI-1, modulate O-GlcNAcylation in rat cortical neurons, and their autophagic effects were determined by immunoblot and immunofluorescence assays. We found that the treatment of cortical neurons with an OGT inhibitor decreased O-GlcNAcylation levels and increased LC3-II expression. Interestingly, the pre-treatment with rapamycin, an mTOR inhibitor, further increased the expression levels of LC3-II induced by OGT inhibition, implicating the involvement of mTOR signaling in O-GlcNAcylation-dependent autophagy. In contrast, OGT inhibitor-mediated autophagy was significantly attenuated by 3-methyladenine (3-MA), a blocker of autophagosome formation. However, when pre-treated with chloroquine (CQ), a lysosomotropic agent and a late-stage autophagy inhibitor, OGT inhibitors significantly increased LC3-II levels along with LC3 puncta formation, indicating the stimulation of autophagic flux. Lastly, we found that OGT inhibitors significantly decreased the levels of the autophagy substrate p62/SQSTM1 while increasing the expression of lysosome-associated membrane protein 1 (LAMP1). Together, our study reveals that the modulation of O-GlcNAcylation by OGT inhibition regulates mTOR-dependent autophagy in rat cortical neurons.

Costunolide Induces Autophagy and Apoptosis by Activating ROS/MAPK Signaling Pathways in Renal Cell Carcinoma.

Although costunolide (Cos), a natural sesquiterpene compound isolated from various medicinal plants, exhibits antiproliferative and pro-apoptotic effects in diverse types of cancers, the mechanism associated with the anticancer property of Cos has not been elucidated. The present investigation was carried out to study the anticarcinogenic influence of Cos on kidney cancer cells. Several human renal cancer cell lines were used and biological and molecular studies were conducted. It was found that Cos significantly suppressed renal carcinoma cell growth via stimulation of apoptosis and autophagy in a concentration-dependent manner. Further studies revealed that Cos increased Bax/Bcl-2 ratio, decreased mitochondrial transmembrane potential (MMP), and enhanced cytoplasmic levels of cytochrome c, and activation of caspase-9, caspase-3, and cleaved PARP, resulting in cell apoptosis. The autophagy induced by Cos resulted from the formation of GFP-LC3 puncta and upregulation of LC3B II and Beclin-1 proteins. Compared with Cos treatment, the autophagy inhibitor 3-MA or ROS scavenger NAC significantly inhibited apoptosis and autophagy. Moreover, NAC and JNK-specific inhibitor SP600125 attenuated the effect of Cos. Taken together, Cos exerted autophagic and apoptotic effects on renal cancer through the ROS/JNK-dependent signal route. These findings suggest that Cos could be a beneficial anticarcinogenic agent.