LC3 Cleavage Research Articles

Marine-Derived Natural Product HDYL-GQQ-495 Targets P62 to Inhibit Autophagy.

Autophagy is widely implicated in pathophysiological processes such as tumors and metabolic and neurodegenerative disorders, making it an attractive target for drug discovery. Several chemical screening approaches have been developed to uncover autophagy-modulating compounds. However, the modulation capacity of marine compounds with significant pharmacological activities is largely unknown. We constructed an EGFPKI-LC3B cell line using the CRISPR/Cas9 knock-in strategy in which green fluorescence indicated endogenous autophagy regulation. Using this cell line, we screened a compound library of approximately 500 marine natural products and analogues to investigate molecules that altered the EGFP fluorescence. We identified eight potential candidates that enhanced EGFP fluorescence, and HDYL-GQQ-495 was the leading one. Further validation with immunoblotting demonstrated that cleaved LC3 was increased in dose- and time-dependent manners, and the autophagy adaptor P62 showed oligomerization after HDYL-GQQ-495 treatment. We also demonstrated that HDYL-GQQ-495 treatment caused autophagy substrate aggregation, which indicated that HDYL-GQQ-495 serves as an autophagy inhibitor. Furthermore, HDYL-GQQ-495 induced Gasdermin E (GSDME) cleavage and promoted pyroptosis. Moreover, HDYL-GQQ-495 directly combined with P62 to induce P62 polymerization. In P62 knockout cells, the cleavage of LC3 or GSDME was blocked after HDYL-GQQ-495 treatment. The EGFPKI-LC3B cell line was an effective tool for autophagy modulator screening. Using this tool, we found a novel marine-derived compound, HDYL-GQQ-495, targeting P62 to inhibit autophagy and promote pyroptosis.

The lectin DrfL inhibits cell migration, adhesion and triggers autophagy-dependent cell death in glioma cells.

Glioblastoma multiforme (GBM) is the most aggressive type of glioma, displaying atypical glycosylation pattern that may modulate signaling pathways involved in tumorigenesis. Lectins are glycan binding proteins with antitumor properties. The present study was designed to evaluate the antitumor capacity of the Dioclea reflexa lectin (DrfL) on glioma cell cultures. Our results demonstrated that DrfL induced morphological changes and cytotoxic effects in glioma cell cultures of C6, U-87MG and GBM1 cell lines. The action of DrfL was dependent upon interaction with glycans, and required a carbohydrate recognition domain (CRD), and the cytotoxic effect was apparently selective for tumor cells, not altering viability and morphology of primary astrocytes. DrfL inhibited tumor cell migration, adhesion, proliferation and survival, and these effects were accompanied by activation of p38MAPK and JNK (p46/54), along with inhibition of Akt and ERK1/2. DrfL also upregulated pro-apoptotic (BNIP3 and PUMA) and autophagic proteins (Atg5 and LC3 cleavage) in GBM cells. Noteworthy, inhibition of autophagy and caspase-8 were both able to attenuate cell death in GBM cells treated with DrfL. Our results indicate that DrfL cytotoxicity against GBM involves modulation of cell pathways, including MAPKs and Akt, which are associated with autophagy and caspase-8 dependent cell death.

Inhibition of Carbonic Anhydrase 2 Overcomes Temozolomide Resistance in Glioblastoma Cells.

About 95% of Glioblastoma (GBM) patients experience tumor relapse as a consequence of resistance to the first-line standard chemotherapy using temozolomide (TMZ). Recent studies reported consistently elevated expression levels of carbonic anhydrase CA2 in recurrent glioblastoma and temozolomide-resistant glioblastoma stem-like cells (GSCs). Here we show that CA2 is preferentially expressed in GSCs and upregulated by TMZ treatment. When expressed in GBM cell lines, CA2 exerts significant metabolic changes reflected by enhanced oxygen consumption and increased extracellular acidification causing higher rates of cell invasion. Notably, GBM cells expressing CA2 respond to combined treatment with TMZ and brinzolamide (BRZ), a non-toxic and potent CA2 inhibitor. Interestingly, brinzolamide was more effective than the pan-CA inhibitor Acetazolamide (ACZ) to sensitize naïve GSCs and TMZ-resistant GSCs to TMZ induced cell death. Mechanistically, we demonstrated that the combined treatment of GBM stem cells with TMZ and BRZ caused autophagy of GBM cell lines and GSCs, reflected by enhanced LC3 cleavage (LC3-II) and p62 reduction. Our findings illustrate the potential of CA2 as a chemo-sensitizing drug target in recurrent GBM and propose a combined treatment of TMZ with CA2 inhibitor to tackle GBM chemoresistance and recurrence.

Mineral trioxide aggregate-induced AMPK activation stimulates odontoblastic differentiation of human dental pulp cells.

To investigate the role of autophagy in MTA-induced odontoblastic differentiation of human dental pulp cells (HDPCs). In MTA-treated HDPCs, odontoblastic differentiation was assessed based on expression levels of dentine sialophosphoprotein (DSPP) and dentine matrix protein 1 (DMP1), alkaline phosphatase activity (ALP) activity by ALP staining and the formation of mineralized nodule by Alizarin red S staining. Expression of microtubule-associated protein 1A/1B-light chain3 (LC3), adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signalling molecules and autophagy-related genes was analysed by Western blot analysis and Acridine orange staining was used to detect autophagic lysosome. For in vivo experiments, tooth cavity preparation models on rat molars were established and the expression of proteins-related odontogenesis and autophagy markers was observed by Immunohistochemistry and Western blot analysis. Kruskal-Wallis with Dunn's multiple comparison was used for statistical analysis. Mineral trioxide aggregate (MTA) promoted odontoblastic differentiation of HDPCs, accompanied by autophagy induction, including formation of autophagic lysosome and cleavage of LC3 to LC3II (P<0.05). Conversely, inhibition of autophagy through 3MA significantly attenuated the expression level of DSPP (P<0.05) and DMP1 (P<0.05) as well as formation of mineralized nodules (P<0.05), indicating the functional significance of autophagy in MTA-induced odontoblastic differentiation. Also, MTA increased the activity of AMPK (P<0.01), whereas inhibition of AMPK by compound C downregulated DSPP (P<0.01) and DMP1 (P<0.05), but increased the phosphorylation of mTOR (P<0.05), p70S6 (P<0.01) and Unc-51-like kinases 1 (ULK1) (ser757) (P<0.01), explaining the involvement of AMPK pathway in MTA-induced odontoblast differentiation. In vivo study, MTA treatment after tooth cavity preparation on rat molars upregulated DMP-1 and DSPP as well as autophagy-related proteins LC3II and p62, and enhanced the phosphorylation of AMPK. MTA induced odontoblastic differentiation and mineralization by modulating autophagy with AMPK activation in HDPCs. Autophagy regulation is a new insight on regenerative endodontic therapy using MTA treatment.

ConBr lectin modulates MAPKs and Akt pathways and triggers autophagic glioma cell death by a mechanism dependent upon caspase-8 activation

Glioblastoma multiforme is the most aggressive type of glioma, with limited treatment and poor prognosis. Despite some advances over the last decade, validation of novel and selective antiglioma agents remains a challenge in clinical pharmacology. Prior studies have shown that leguminous lectins may exert various biological effects, including antitumor properties. Accordingly, this study aimed to evaluate the mechanisms underlying the antiglioma activity of ConBr, a lectin extracted from the Canavalia brasiliensis seeds. ConBr at lower concentrations inhibited C6 glioma cell migration while higher levels promoted cell death dependent upon carbohydrate recognition domain (CRD) structure. ConBr increased p38MAPK and JNK and decreased ERK1/2 and Akt phosphorylation. Moreover, ConBr inhibited mTORC1 phosphorylation associated with accumulation of autophagic markers, such as acidic vacuoles and LC3 cleavage. Inhibition of early steps of autophagy with 3-methyl-adenine (3-MA) partially protected whereas the later autophagy inhibitor Chloroquine (CQ) had no protective effect upon ConBr cytotoxicity. ConBr also augmented caspase-3 activation without affecting mitochondrial function. Noteworthy, the caspase-8 inhibitor IETF-fmk attenuated ConBr induced autophagy and C6 glioma cell death. Finally, ConBr did not show cytotoxicity against primary astrocytes, suggesting a selective antiglioma activity. In summary, our results indicate that ConBr requires functional CRD lectin domain to exert antiglioma activity, and its cytotoxicity is associated with MAPKs and Akt pathways modulation and autophagy- and caspase-8- dependent cell death.

Abstract 4170: Decursin inhibits gastric cancer cell growth and autophagic flux by suppressing cathepsin C

Abstract Purpose: Autophagy is a series of processes in which biomolecules are digested and recycled, and it also provides energy for the development and progression of cancer. Thus, autophagy inhibitors can be used to suppress the progression of cancer. In this study, we investigated whether decursin, which is derived from natural products, inhibits the growth and autophagy of cancer cells. Methods: The gastric cancer cell lines SNU216 and NCI-N87 were treated with or without decursin. Cell growth was evaluated by Cell-Counting Kit-8, colony formation, and BrdU assays, as well as by propidium iodide staining, and autophagic flux was evaluated by western blotting, LC3 puncta, GFP-RFP-LC3, and transmission electron microscopy. Finally, tumor spheroids and patient-derived gastric cancer organoids were used to confirm the effect of decursin in three-dimensional culture. Results: Decursin reduced the growth and anchorage-dependent viability of gastric cancer cells. LC3 cleavage and LC3-GFP puncta were increased by decursin in a time- and concentration-dependent manner. However, the increase in autophagosome formation did not lead to an increase in autophagic flux. Decursin downregulated cathepsin C (CTSC), a lysosomal protease, irrespective of cellular pH. In addition, knockdown of CTSC reduced the mRNA and protein levels of E2F3, resulting in cell-cycle arrest. Decursin also reduced cell growth in both tumor spheroids and patient-derived gastric cancer organoids. Conclusion: The results demonstrated that decursin inhibited tumor growth and progression by suppressing CTSC-mediated autophagic flux and cell-cycle progression. Therefore, decursin is a novel inhibitor of autophagic flux, with potential for the treatment of gastric cancer. Citation Format: Solbi Kim, Mina Joo, Nayoung KIM, Myung-Won Lee, Heung Jin Jeon, Hyewon Ryu, Ik-Chan Song, Hyo Jin Lee. Decursin inhibits gastric cancer cell growth and autophagic flux by suppressing cathepsin C [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4170.

Autophagy interferes with human cytomegalovirus genome replication, morphogenesis, and progeny release

ABSTRACT Viral infections are often accompanied by the induction of autophagy as an intrinsic cellular defense mechanism. Herpesviruses have developed strategies to evade autophagic degradation and to manipulate autophagy of the host cells to their benefit. Here we addressed the role of macroautophagy/autophagy in human cytomegalovirus replication and for particle morphogenesis. We found that proteins of the autophagy machinery localize to cytoplasmic viral assembly compartments and enveloped virions in the cytoplasm. Surprisingly, the autophagy receptor SQSTM1/p62 was also found to colocalize with HCMV capsids in the nucleus of infected cells. This finding indicates that the autophagy machinery interacts with HCMV already at the early nuclear stages of particle morphogenesis. The membrane-bound form of LC3 and several autophagy receptors were packaged into extracellular HCMV virions. This suggested that autophagic membranes were included during secondary envelopment of HCMV virions. To further address the importance of autophagy in HCMV infection, we generated an HCMV mutant that expressed a dominant-negative version of the protease ATG4B (BAD-ATG4BC74A). The proteolytic activity of ATG4B is required for LC3 cleavage, priming it for membrane conjugation. Surprisingly, both genome replication and virus release were enhanced in cells infected with BAD-ATG4BC74A, compared to control strains. These results show that autophagy operates as an antiviral process during HCMV infection but is dispensable for secondary HCMV particle envelopment. Abbreviations: ATG: autophagy-related; BAC: bacterial artificial chromosome; BECN1: beclin 1; CPE: cytopathic effect; cVACs: cytoplasmic viral assembly compartments; d.p.i.: days post-infection; DB: dense body; EBV: Epstein-Barr virus; galK: galactokinase; HCMV: human cytomegalovirus; HFF: human foreskin fibroblasts; IE: immediate-early; IRS: internal repeat short; LC3: MAP1LC3A/B; m.o.i.; multiplicity of infection; MCP: major capsid protein; Pp: phosphoprotein; sCP/UL48a: smallest capsid protein; TRS: terminal repeat short; UL: unique long; US: unique short

Novel Atg4B inhibitors potentiate cisplatin therapy in lung cancer cells through blockade of autophagy

Autophagy induction in tumors is implicated in tumor malignancy and acquisition of anticancer-drug resistance, and hence, autophagy inhibitors are expected as novel anticancer agents. Atg4B, a cysteine proteinase, is a key enzyme in autophagy process, and considered as one of desirable targets for development of selective autophagy inhibitors. In this study, to search novel inhibitors of Atg4B, we employed the virtual screening approach and differential scanning fluorimetry analysis, and found two compounds 1 (2-methyl-5-oxo-5H-[1,3,4]thiadiazolo-[3,2-a]pyrimidine-6-carboxylic acid) and 17 (methyl 4-({2-[(4,6-diaminopyrimidin-2-yl)sulfanyl]propanoyl} amino)benzoate), showing IC50 values of 10 and 12 µM, respectively, in in vitro LC3 cleavage assay of Atg4B. Molecular docking analysis suggested that 1 and 17 bind to the catalytic domain of Atg4B. The treatments with 1 and 17 significantly suppressed autophagy induction to lung cancer A549 cells by tamoxifen and cisplatin, in which their cotreatments promoted tamoxifen- and cisplatin-induced apoptotic cell death. Moreover, the autophagy suppression by 1 and 17 remarkably restored the sensitivity to cisplatin in cisplatin-resistant A549 cells. Thus, 1 and 17 represent promising leads for future development of potent and selective Atg4B inhibitors that augment existing chemotherapy efficacy.

LncRNA KCNQ1OT1 enhances the chemoresistance of oxaliplatin in colon cancer by targeting the miR-34a/ATG4B pathway.

PurposeThe chemoresistance of colon cancer to oxaliplatin (L-OHP) indicates poor prognosis. Long non-coding RNA (lncRNA) KCNQ1OT1 (KCNQ1 opposite strand/antisense transcript 1) has been shown to participate in the tumorigenesis of several types of cancers. However, little is known about the role of KCNQ1OT1 in the chemoresistance and prognosis of colon cancer.Materials and methodsQuantitative-PCR and Western blot were used to measure the expression profiles of KCNQ1OT1, miR-34a, and Atg4B in colon cancer tissues and cells. Cell viability assay and flow cytometry were used to examine their effects on cell proliferation and death. Cleavage of LC3 and GFP-LC3 plasmid transfection were used to detect autophagic activity. Double luciferase reporter assay was used to verify the interactions between miRNA and lncRNA or mRNA. Xenograft tumor model was used to verify the effects of KCNQ1OT1 in vivo.ResultsIn this study, it is shown that the expression level of KCNQ1OT1 was increased in tumor, which indicated poor prognosis in colon cancer patients. Using colon cancer cell lines HCT116 and SW480, it was demonstrated that knockdown of KCNQ1OT1 decreased the cell viability and increased the apoptosis rates upon L-OHP treatment. Further studies indicated that Atg4B upregulation was partially responsible for KCNQ1OT1-induced protective autophagy and chemoresistance. Moreover, miR-34a functioned as a bridge between KCNQ1OT1 and Atg4B, which could be sponged by KCNQ1OT1, while it could also bind to the 3′-UTR of Atg4B and downregulate its expressions. Finally, we show that the KCNQ1OT1/miR-34a/Atg4B axis regulated the chemoresistance of colon cancer cells in vitro and in vivo.ConclusionlncRNA KCNQ1OT1 promoted the chemoresistance of colon cancer by sponging miR-34a, thus upregulating the expressions of Atg4B and enhancing protective autophagy. KCNQ1OT1 might become a promising target for colon cancer therapeutics.

Down-regulated TMED10 in Alzheimer disease induces autophagy via ATG4B activation

ABSTRACTSeveral studies have shown that dysfunction of macroautophagy/autophagy is associated with many human diseases, including neurodegenerative disease and cancer. To explore the molecular mechanisms of autophagy, we performed a cell-based functional screening with SH-SY5Y cells stably expressing GFP-LC3, using an siRNA library and identified TMED10 (transmembrane p24 trafficking protein 10), previously known as the γ-secretase-modulating protein, as a novel regulator of autophagy. Further investigations revealed that depletion of TMED10 induced the activation of autophagy. Interestingly, protein-protein interaction assays showed that TMED10 directly binds to ATG4B (autophagy related gene 4B cysteine peptidase), and the interaction is diminished under autophagy activation conditions such as rapamycin treatment and serum deprivation. In addition, inhibition of TMED10 significantly enhanced the proteolytic activity of ATG4B for LC3 cleavage. Importantly, the expression of TMED10 in AD (Alzheimer disease) patients was considerably decreased, and downregulation of TMED10 increased amyloid-β (Aβ) production. Treatment with Aβ increased ATG4B proteolytic activity as well as dissociation of TMED10 and ATG4B. Taken together, our results suggest that the AD-associated protein TMED10 negatively regulates autophagy by inhibiting ATG4B activity.Abbreviations: Aβ: amyloid-β; AD: Alzheimer disease; ATG: autophagy related; BECN1: beclin 1; BiFC: bimolecular fluorescence complementation; CD: cytosolic domain; GFP: green fluorescent protein; GLUC: Gaussia luciferase; IP: immunoprecipitation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LD: luminal domain; PD: Parkinson disease; ROS: reactive oxygen species; siRNA: small interfering RNA; SNP: single-nucleotide polymorphisms; TD: transmembrane domain; TMED10: transmembrane p24 trafficking protein 10; VC: C terminus of Venus fluorescent protein; VN: N terminus of Venus fluorescent protein.

Anti-glioma properties of DVL, a lectin purified from Dioclea violacea

Plant lectins have been studied owing to their structural properties and biological effects that include agglutinating activity, antidepressant-like effect and antitumor property. The results from this work showed the effects of the lectin extracted from the Dioclea violacea plant (DVL) on the C6 rat glioma cell line. DVL treatment was able to induce caspase-3 activation, apoptotic cell death and cellular membrane damage. Furthermore, DVL decreased mitochondrial membrane potential and increased the number of acidic vesicles and cleavage of LC3, indicating activation of autophagic processes. DVL also significantly inhibited cell migration. Compared to ConA, a well-studied lectin extracted from Canavalia ensiformes seeds, some effects of DVL were more potent, including decreasing C6 glioma cell viability and migration ability. Taken together, the results suggest that DVL can induce glioma cell death, autophagy and inhibition of cell migration, displaying potential anti-glioma activity.

ATG4B contains a C-terminal LIR motif important for binding and efficient cleavage of mammalian orthologs of yeast Atg8

ABSTRACTThe cysteine protease ATG4B cleaves off one or more C-terminal residues of the inactive proform of proteins of the ortholog and paralog LC3 and GABARAP subfamilies of yeast Atg8 to expose a C-terminal glycine that is conjugated to phosphatidylethanolamine during autophagosome formation. We show that ATG4B contains a C-terminal LC3-interacting region (LIR) motif important for efficient binding to and cleavage of LC3 and GABARAP proteins. We solved the crystal structures of the GABARAPL1-ATG4B C-terminal LIR complex. Analyses of the structures and in vitro binding assays, using specific point mutants, clearly showed that the ATG4B LIR binds via electrostatic-, aromatic HP1 and hydrophobic HP2 pocket interactions. Both these interactions and the catalytic site-substrate interaction contribute to binding between LC3s or GABARAPs and ATG4B. We also reveal an unexpected role for ATG4B in stabilizing the unlipidated forms of GABARAP and GABARAPL1. In mouse embryonic fibroblast (MEF) atg4b knockout cells, GABARAP and GABARAPL1 were unstable and degraded by the proteasome. Strikingly, the LIR motif of ATG4B was required for stabilization of the unlipidated forms of GABARAP and GABARAPL1 in cells.

Involvement of Endoplasmic Reticulum Stress, Autophagy, and Apoptosis in Advanced Glycation End Products-Induced Glomerular Mesangial Cell Injury.

Advanced glycation end-products (AGEs)-induced mesangial cell death is one of major causes of glomerulus dysfunction in diabetic nephropathy. Both endoplasmic reticulum (ER) stress and autophagy are adaptive responses in cells under environmental stress and participate in the renal diseases. The role of ER stress and autophagy in AGEs-induced mesangial cell death is still unclear. Here, we investigated the effect and mechanism of AGEs on glomerular mesangial cells. AGEs dose-dependently decreased mesangial cell viability and induced cell apoptosis. AGEs also induced ER stress signals in a time- and dose-dependent manner. Inhibition of ER stress with 4-phenylbutyric acid effectively inhibited the activation of eIF2α and CHOP signals and reversed AGEs-induced cell apoptosis. AGEs also activated LC-3 cleavage, increased Atg5 expression, and decreased p62 expression, which indicated the autophagy induction in mesangial cells. Inhibition of autophagy by Atg5 siRNAs transfection aggravated AGEs-induced mesangial cell apoptosis. Moreover, ER stress inhibition by 4-phenylbutyric acid significantly reversed AGEs-induced autophagy, but autophagy inhibition did not influence the AGEs-induced ER stress-related signals activation. These results suggest that AGEs induce mesangial cell apoptosis via an ER stress-triggered signaling pathway. Atg5-dependent autophagy plays a protective role. These findings may offer a new strategy against AGEs toxicity in the kidney.

O-GlcNAcylation of ATG4B positively regulates autophagy by increasing its hydroxylase activity.

Autophagy is a catabolic degradation process and maintains cellular homeostasis. And autophagy is activated in response to various stress conditions. Although O-GlcNAcylation functions a sensor for nutrient and stress, the relationship between O-GlcNAcylation and autophagy is largely unknown. Here, we identified that ATG4B is novel target for O-GlcNAcylation under metabolic stress condition. Treatment with PugNAc, an O-GlcNAcase inhibitor increased activation of autophagy in SH-SY5Y cells. Both bimolecular fluorescence complementation and immunoprecipitation assay indicated that OGT directly interacts with ATG4B in SH-SY5Y cells. We also found that the O-GlcNAcylated ATG4B was increased in autophagy activation conditions, and down-regulation of OGT reduces O-GlcNAcylation of ATG4B under low glucose condition. Furthermore, the proteolytic activity of ATG4B for LC3 cleavage was enhanced in PugNAc-treated cells. Taken together, these results imply that O-GlcNAcylation of ATG4B regulates autophagy activation by increasing its proteolytic activity under metabolic stress condition.

Abstract 1241: Podophyllotoxin acetate (PA) as an anti-cancer drug candidate regulates various malignancy traits of non-small cell lung cancer

Abstract Here, we reported that podophyllotoxin acetate (PA) showed various aniti-cancer effects: induction of several cell death pathways, inhibition of γ-ionizing radiation (IR)-induced cancer invasion, chemo-sensitizer effect with topoisomerase (TOP) inhibitors against non-small cell lung cancer (NSCLC) cell lines as follows. (1) Stimulation of cell death via various mechanisms. First, PA dose-dependently induced cell cycle arrest at G2/M phase, as shown by accumulation of the mitosis-related proteins, p21, survivin and aurora B. This arrest was due to the PA-induced inhibition of microtubule polymerization that related to DNA damage (reflected by accumulation of γ-H2AX) resulting induction of intrinsic/extrinsic damage apoptotic pathways. PA also activated the pro-apoptotic ER stress pathway, as evidenced by increased expression levels of BiP, CHOP, IRE1-a, phospho-PERK, and phospho-JNK. Next, PA induced autophagy that reflected the expression of beclin-1, Atg3, Atg5 and Atg7, and the cleavage of LC3. (2) Inhibition of IR-induced cancer invasion. IR increased the invasion/migration of A549 cells, and this effect was decreased by PA treatment that the expressions/activities of matrix metalloprotase (MMP) -2, MMP-9, and vimentin, suggesting that PA could block the IR-induced epithelial-mesenchymal transition (EMT). In this study, we also identified IR-induced new signaling pathway, EGFR - p38/ERK - CREB-1/STAT3 - EMT pathway, that was inhibited by PA. (3) Chemo-sensitizer effect conjugated with TOP inhibitors - etoposide (Eto) and camptothecin (Cpt). Combination Index (CI) values of the combinations showed synergistic effects between PA and topoisomerase inhibitors. Combination with PA and Eto/Cpt promotes disruption of dynamics of actin filaments, which subsequently enhanced apoptotic cell death that accompanied to increased phosphorylation of p38. We also observed these combinations also inhibited activation of CREB-1, synergistically. Therefore, this study indicated that PA functions as a chemosensitizer by enhancing apoptosis through activation of p38/caspases apoptotic axis and suppression of CREB-1.Taken together; these results suggest PA is a new anti-cancer drug candidate that could control several malignancy traits of cancer including uncontrolled cell cycle and IR-induced invasion. Citation Format: Jeong Hyun Cho, Wan Gi Hong, Sang-Gu Hwang, Hong-Duck Um, Jong Kuk Park. Podophyllotoxin acetate (PA) as an anti-cancer drug candidate regulates various malignancy traits of non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1241.

Abstract 2603: Discovery of chemopreventive agents from licorice (Glycyrrhiza Uralensis)

Abstract Traditional Chinese Medicine (TCM) provides a rich resource of potential chemoprevention agents; however, the complexity arise from its multi-components and multi-targets nature hindered in-depth investigations and translations. Licorice is one of the most commonly utilized TCM herbal medicine which possesses many pharmacological activities including cancer prevention. We isolated over 120 licorice-derived compounds and systemically screened these compounds using cellular and molecular models including cytotoxicity, nitric oxide production, NF-κB and ARE luciferase reporters, and other models. Isoangustone A (IAA) is an isoprenyled isoflavonoid which has been reported to induce apoptosis and cell cycle arrest in prostate cancer cells, and it was identified as a NF-κB inhibitor in our assays. IAA significantly inhibited NF-κB signaling in SW480 colorectal adenocarcinoma cells, and decreased the expression of Cox-2 and iNOS. Overexpression of constitutively activated IKKβ potently stimulated NF-κB signaling and reversed IAA-mediated inhibition. Oral administration of IAA significantly inhibited dextran sodium sulfate-induced NF-κB signaling and the expression of Cox-2 and iNOS in mouse colon tissue, and alleviated symptoms of colitis without observable acute toxicity. IAA and similar compounds also induced autophagy in various cancer cells, and inhibition of autophagy significant reversed IAA-induced cell death, suggesting the existence of autophagic cell death. IAA caused cleavage and aggregation of LC-3 accompanied with p62 degradation, and typical autophagic morphology was observed by transmission electronic microscopy. Overexpression of dominant negative AMPK or treatment with AMPK inhibitor demonstrated that activation of AMPK was important for IAA-induced autophagy, while inhibition of Akt was not. Further studies suggest that metabolic stresses is essential for the pharmacological activities of IAA, and primary molecular target were identified. Notably, IAA significantly inhibited tumor growth in nude mice xenograft model. These results suggest that IAA is potentially an anti-inflammatory bowel disease and anti- colorectal cancer agent. Based on the structural similarity, several other compounds were found to possess even stronger anti-inflammation, pro-apoptotic and autophagy-inducing activities. The present work discovered several promising chemopreventive compounds from licorice that deserve future study, and provides an example of study on TCM in chemoprevention. Several other compounds were identified as Nrf2/ARE signaling modulators or apoptosis/autophagy inducers and further investigated. The present works were supported by the National Natural Science Foundation of China (No. 81272468, 81472657). Citation Format: Shunan Tang, Shuai Ji, Yongrui Wang, Ye Min, Siwang Yu, Tony A-N. Kong. Discovery of chemopreventive agents from licorice (Glycyrrhiza Uralensis). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2603.

Angiostatic actions of capsicodendrin through selective inhibition of VEGFR2-mediated AKT signaling and disregulated autophagy.

Angiogenesis is the formation of new blood vessels from existing vasculature critical for embryonic development and vascular remodeling. Its dysregulation underlies numerous pathologic states ranging from ischemia to tumor growth and as such identifying new targeted- therapies is of significant interest for angiogenesis-based medicine. Here we evaluated the potential angiostatic properties of capsicodendrin (CPCD), a natural compound isolated from Cinnamosma macrocarpa, a plant belonging to the Malagasy Cinnamosma. CPCD potently inhibits endothelial proliferation, migration and capillary tube formation at nanomolar to low micromolar concentrations without inducing cytotoxic effects. We show that CPCD directly inactivates VEGFR2 and downstream AKT signaling, thereby strongly inducing autophagy as determined by increased expression of beclin1, autophagy-related gene (Atg) 3, Atg5 and LC3 cleavage. Ectopic AKT overexpression counteracts the inhibitory effects of CPCD on proliferation and capillary tubule formation. Importantly, CPCD treatment in vivo inhibits sprouting angiogenesis as evidenced by strongly reduced intersegmental vessel (ISV) sprouting and subintestinal vessel (SIV) formation during zebrafish embryonic development, and correlates with increased presence of LC3II along the ISVs despite overall reduced vasculature. These findings demonstrate CPCD as a potent inhibitor of the VEGFR2/AKT pathway at nanomolar concentrations and inducer of autophagy-related angiostatic effects.

The BH3 mimetic drug ABT-737 induces apoptosis and acts synergistically with chemotherapeutic drugs in thyroid carcinoma cells.

BackgroundPatients with dedifferentiated and anaplastic thyroid carcinomas that do not take up radioiodine are resistant to chemotherapeutic treatment and external irradiation and thus are difficult to treat. Direct induction of apoptosis is a promising approach in these apoptosis-resistant tumor cells. The BH3 mimetic ABT-737 belongs to a new class of drugs that target anti-apoptotic proteins of the BCL-2 family and facilitate cell death. The purpose of this study was to investigate the effect of ABT-737 alone or in combination with chemotherapeutic drugs on thyroid carcinoma cell lines.MethodsA total of 16 cell lines derived from follicular, papillary, and anaplastic thyroid carcinomas were treated with ABT-737. Cell viability was measured with MTT assay. Cell death was determined by cell cycle phase distribution and subG1 peak analyses, determination of caspase 3/7 activity and caspase cleavage products, lactate dehydrogenase (LDH) liberation assays and LC3 analysis by western blot.ResultsThe number of viable cells was decreased in all cell lines examined after ABT-737 treatment, with IC50 values ranging from 0.73 to 15.6 μM. Biochemical markers of apoptosis like caspase activities, caspase cleavage products and DNA fragmentation determined as SubG1 peak were elevated after ABT-737 treatment, but no LC3 cleavage was induced by ABT-737 indicating no autophagic processes. In combination with doxorubicin and gemcitabine, ABT-737 showed synergistic effects on cell viability.ConclusionsWith these experiments we demonstrated the efficacy of the BH3 mimetic drug ABT-737 against dedifferentiated thyroid carcinoma cells of various histological origins and showed synergistic effects with chemotherapeutic drugs. ABT-737-treated cells underwent an apoptotic cell death. ABT-737 and related BH3 mimetic drugs, alone or in combination, may thus be of value as a new therapeutic option for dedifferentiated thyroid carcinomas.

Autophagy Prevents Oxidative Stress-Induced Loss of Self-Renewal Capacity and Stemness in Human Tendon Stem Cells by Reducing ROS Accumulation

Background/Aims: Tendon stem cells (TSCs) exhibit a high self-renewal capacity, multi-differentiation potential, and low immunogenicity; thus, these cells might provide a new cell source for tendon repair and regeneration. TSCs are exposed to increased oxidative stress at tendon injury sites; however, how TSCs maintain their stemness under oxidative stress is not clear. Methods and Results: In this study, we found that H₂O₂ treatment increased ROS accumulation in human TSCs (hTSCs) and resulted in loss of self-renewal capacity and stemness, as reflected in reduced colony formation and proliferation, decreased expression of the stemness markers Nanog, Oct-4, NS, and SSEA-4, and impaired differentiation capability. These H₂O₂-induced damages were prevented by pretreatment with starvation or rapamycin. Pretreatment with starvation or rapamycin prior to H₂O₂ exposure also led to decreased intracellular and mitochondrial ROS accumulation along with increased autophagic activity, as manifested in increased LC3 cleavage, Beclin-1 expression, and GFP-LC3-labeled autophagosome formation. Autophagy inhibition by 3-MA or CQ, or by shRNA silencing of Agt-7 or Beclin-1 reduced the protective effects of starvation and rapamycin on H₂O₂-treated hTSCs. Conclusion: Thus, the findings of this study suggest that autophagy prevents oxidative stress-induced loss of self-renewal capacity and stemness in hTSCs through suppression of ROS accumulation.

Isoalantolactone induces autophagic cell death in SKOV₃ human ovarian carcinoma cells via upregulation of PEA-15.

We investigated the effects of isoalantolactone on cell growth inhibition and underlying cell death mechanisms in SKOV3 human ovarian cancer cells. The effects of isoalantolactone on cell proliferation and cell cycle were examined by EdU incorporation assay and DNA content assay. Western blotting was performed to determine the protein expression effects of isoalantolactone on cell cycle‑related proteins, autophagic regulators and PEA‑15. Autophagic vacuoles were observed by acridine orange staining. PEA‑15 knockdown by siRNA was used to confirm that PEA‑15 was involved in isoalantolactone‑induced autophagy of SKOV3 cells. Isoalantolactone inhibited the viability and proliferation of SKOV3 cells in a dose‑ and time‑dependent fashion. Isoalantolactone induced cell cycle arrest at G2/M phase and decreased the expression of cell cycle‑related proteins cyclin B1 and CDK1 in SKOV3 cells. Accordingly, isoalantolactone also induced SKOV3 cell autophagy via accumulation of autophagic vacuoles in the cytoplasm, increased Beclin1 protein expression, and increased LC3 cleavage. Furthermore, we observed that isoalantolactone‑induced autophagy was through increased PEA‑15 expression and the phosphorylation of ERK, whereas less change was observed to autophagy on SKOV3 cells through PEA‑15 knockdown by siRNA. Isoalantolactone‑induced autophagic cell death was further confirmed by pretreatment with the autophagy inhibitor 3‑methyladenine (3‑MA). In conclusion, isoalantolactone induced cell cycle arrest and autophagy and inhibited cell proliferation of SKOV3 cells via the upregulated PEA‑15 expression and the phosphorylation of ERK.