Blockage Of Autophagy Research Articles

ATG5 cancer mutations and alternative mRNA splicing reveal a conjugation switch that regulates ATG12\u2013ATG5-ATG16L1 complex assembly and autophagy

Autophagy is critical for maintaining cellular homeostasis during times of stress, and is thought to play important roles in both tumorigenesis and tumor cell survival. Formation of autophagosomes, which mediate delivery of cytoplasmic cargo to lysosomes, requires multiple autophagy-related (ATG) protein complexes, including the ATG12–ATG5-ATG16L1 complex. Herein, we report that a molecular ATG5 “conjugation switch”, comprised of competing ATG12 and ubiquitin conjugation reactions, integrates ATG12–ATG5-ATG16L1 complex assembly with protein quality control of its otherwise highly unstable subunits. This conjugation switch is tightly regulated by ATG16L1, which binds to free ATG5 and mutually protects both proteins from ubiquitin conjugation and proteasomal degradation, thereby instead promoting the irreversible conjugation of ATG12 to ATG5. The resulting ATG12–ATG5 conjugate, in turn, displays enhanced affinity for ATG16L1 and thus fully stabilizes the ATG12–ATG5-ATG16L1 complex. Most importantly, we find in multiple tumor types that ATG5 somatic mutations and alternative mRNA splicing specifically disrupt the ATG16L1-binding pocket in ATG5 and impair the essential ATG5-ATG16L1 interactions that are initially required for ATG12–ATG5 conjugation. Finally, we provide evidence that ATG16L2, which is overexpressed in several cancers relative to ATG16L1, hijacks the conjugation switch by competing with ATG16L1 for binding to ATG5. While ATG16L2 stabilizes ATG5 and enables ATG12–ATG5 conjugation, this endogenous dominant-negative inhibitor simultaneously displaces ATG16L1, resulting in its proteasomal degradation and a block in autophagy. Thus, collectively, our findings provide novel insights into ATG12–ATG5-ATG16L1 complex assembly and reveal multiple mechanisms wherein dysregulation of the ATG5 conjugation switch inhibits autophagy.

Tubeimoside-I sensitizes colorectal cancer cells to chemotherapy by inducing ROS-mediated impaired autophagolysosomes accumulation

BackgroundTubeimoside-I (TBM), a plant-derived bioactive compound, shows antitumor activity in different tumors and can enhance the efficacy of chemotherapeutic agents. However, the detail mechanism underlying remains to be elucidated.MethodsThe cytotoxic potential of TBM towards CRC cells was examined by CCK8 assay, colony formation, LDH release assay, flow cytometry method and Western blots. The ROS levels, autophagy, apoptosis, chemosensitivity to 5-FU or DOX, etc. were determined between control and TBM-treated CRC cells.ResultsIn this study, we found that TBM could inhibit proliferation and induce apoptosis in colorectal cancer (CRC) cells. Intriguingly, TBM treatment could either promote autophagy initiation by ROS-induced AMPK activation, or block autophagy flux through inhibiting lysosomal hydrolytic enzymes, which leaded to massive impaired autophagylysosomes accumulation. Administration of autophagy initiation inhibitor (3-MA or selective ablation of autophagy related proteins) relieves TBM-induced CRC suppression, while combination use of autophagy flux inhibitor chloroquine (CQ) slightly augments TBM-induced cell death, suggesting that impaired autophagylysosomes accumulation contributes to TBM-induced growth inhibition in CRC cells. Notably, as an autophagy flux inhibitor, TBM works synergistically with 5-fluorouracil (5-FU) or doxorubicin (DOX) in CRC suppression.ConclusionTogether, our study provides new insights regarding the anti-tumor activity of TBM against CRC, and established potential applications of TBM for CRC combination therapies in clinic.

Inhibition of ROS/NUPR1-dependent autophagy antagonises repeated cadmium exposure -induced oral squamous cell carcinoma cell migration and invasion

Cadmium (Cd), an established carcinogen, is a risk factor for oral squamous cell carcinoma (OSCC). Macroautophagy/autophagy is proposed to play a pivotal role in Cd-mediated carcinogenic activity. However, the mechanisms underlying Cd-induced autophagy are poorly understood. In the present study, a CAL27 OSCC cell line exposed to 10−6 M Cd for 8 weeks was used as a model system. Repeated Cd exposure induced significant migration and invasion of CAL27 cells. Furthermore, we showed that Cd increased the autophagic flux in CAL27 cells, as evidenced by the upregulation of LC3-II and the downregulation of P62/SQSTM1. The genetic blocking of autophagy inhibited Cd-induced migration and invasion, indicating a carcinogenic role of autophagy in Cd-treated CAL27 cells. Cd-induced NUPR1 expression, which contributes to lysosomal biogenesis and expression of autophagy-related gene, was found to mechanistically initiate autophagy in CAL27 cells. Of note, NUPR1 shRNA abolished Cd-induced autophagy both in vitro and in vivo. We also found that Cd triggered the generation of MDA in a xenograft tumour model and that N-acetyl-l-cysteine, a reactive oxygen species (ROS) scavenger, abrogated the effects of Cd on NUPR1-dependent autophagy in vivo. Taken together, these results demonstrate that ROS-dependent NUPR1-mediated autophagy plays an important role in repeated Cd exposure -induced cell growth, migration and invasion in OSCC cells.

Abstract 3085: A novel salicylanilide derivative inhibits castration-resistant prostate cancer via endoplasmic reticulum stress-triggered PERK autophagic signaling pathway

Abstract Background and Purpose: Metastatic castration-resistant prostate cancer (CRPC) is currently incurable. This study is to extend pharmacological value of our synthesized salicylanilide derivative, that has previously testified for its osteoclastogenesis activity, by exploring its additional cytotoxic properties and underlying mechanism in CRPC cells. Experimental Approach: LCC03, a 5-(2′,4′-Difluorophenyl)-salicylanilide derivatives, was chemically synthesized and examined for cell growth inhibition in a serial of CRPC cell lines. Possible mechanisms involved in the anticancer activity was elucidated based on the changes in pathway-related protein expression and cellular events, together with antagonists for functional validation. The therapeutic efficacy and safety of LCC03 for the treatment of bone metastatic CRPC was evaluated in an experimental animal model. Key Results: LCC03 dose-dependently suppressed proliferation and retarded cell cycle progression in CRPC cells. The classical autophagy features including autophagsomes formation and LC3-II conversation were dramatically shown in LCC03-treated CRPC cells, and it was associated with the suppressed AKT/mTOR signaling pathways, a major negative regulator of autophagy, Moreover, an expanded morphology of the ER, increased expression of the ER stress markers GRP78 and PERK, and eIF2α phosphorylation were observed. Blockage of autophagy and PERK pathways using small molecule inhibitors or shRNA knockdown reversed LCC03-induced autophagy and cell death, thus indicating that the PERK-eIF2α pathway contributed to the autophagy induced by LCC03. Furthermore, treatment of tumor-bearing mice with intraperitoneal administered LCC03 suppressed the growth of CRPC xenografts in mouse bone without systemic toxicity. Conclusion and Implications: LCC03 induces cellular responses toward ER stress-induced autophagy via the PERK-eIF2α pathway that impairs proliferation and cell cycle progression and exhibits cytotoxicity against CRPC cells. The dual action of 5-(2′,4′-difluorophenyl)-salicylanilide on both the osteoclasts and the tumor cells strongly indicates that LCC03 is a promising anticancer candidate for preventing and treating metastatic CRPC. Citation Format: Chia-Ling HSIEH, Hsu-Shan Hung, Kuan-Chou Chen, Teigi Saka, Chih-Ying Chiang, Shian-Ying Sung. A novel salicylanilide derivative inhibits castration-resistant prostate cancer via endoplasmic reticulum stress-triggered PERK autophagic signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3085.

Combining use of Phillyrin and autophagy blocker alleviates laryngeal squamous cell carcinoma via AMPK/mTOR/p70S6K signaling.

Phillyrin (PHN), one of the major active constituents of Forsythia suspensa and F. koreana, has been reported to produce antioxidant, antibacterial, anti-obesity and anti-inflammatory effects. However, no study has demonstrated the role of PHN in laryngeal squamous cell carcinoma (LSCC). We aimed to investigate the effects of PHN on the proliferation and apoptosis of HEp-2 cells. In the present study, PHN alone showed little effect on HEp-2 cell proliferation and apoptosis. Subsequent tests showed that PHN could largely enhance the level of autophagy on HEp-2 cells. Combining use of PHN and autophagy blockers including 3-methyladenine (3-MA) and chloroquine (CQ) significantly inhibited HEp-2 cell proliferation in a dose- and time-dependent manner and induced apoptosis after 24 h in a dose-dependent manner. Additionally, we found that the possible underlying molecular mechanism of PHN-induced autophagy might be through the AMPK/mTOR/p70S6K signaling pathway. Taken together, our study indicates that combining use of PHN and autophagy blockers may serve as a novel strategy in LSCC treatment.

Human papillomavirus 16E6/E7 activates autophagy via Atg9B and LAMP1 in cervical cancer cells.

BackgroundsAlthough the role of high‐risk human papillomavirus (HPV) E6 and E7 in cellular malignant transformation has been elucidated， the function of both genes in cellular homeostasis is still unknown. Autophagy functions in maintenance of cellular homeostasis play a key role in the initiation and development of cancer and infectious disease.MethodsCervical cancer cell lines SiHa and CaSki were utilized in this study.ResultsWe found that HPV 16E6/E7 (16E6/E7) downregulation inhibited autophagy, and consequently suppressed cell proliferation and promoted early apoptosis. Transcriptome sequencing demonstrated that Atg9B and LAMP1 were downregulated in 16E6/E7 knockdown cells. Gene function experiments revealed that 16E6/E7 downregulation depressed Atg9B and LAMP1, and Atg9B and LAMP1 overexpression compensated, at least partially, autophagy blockage induced by 16E6/E7 knockdown. Immunoprecipitation assay showed that 16E7 interacted with Atg9B and dual‐luciferase reporter system revealed that 16E6 most likely regulated −1750 to −2000 nt in Atg9B and −1800 to −2000 nt in LAMP1 promoter region.ConclusionsOur findings verified that 16E6/E7 activated autophagy via accelerating autophagosome formation and degradation, and Atg9B and LAMP1 were involved in the process of 16E6/E7 modulating autophagy, suggesting that targeting autophagy may be a potential approach in cervical cancer therapeutics.

Inactivation of MTOR promotes autophagy-mediated epithelial injury in particulate matter-induced airway inflammation

ABSTRACT Particulate matter (PM) is able to induce airway epithelial injury, while the detailed mechanisms remain unclear. Here we demonstrated that PM exposure inactivated MTOR (mechanistic target of rapamycin kinase), enhanced macroautophagy/autophagy, and impaired lysosomal activity in HBE (human bronchial epithelial) cells and in mouse airway epithelium. Genetic or pharmaceutical inhibition of MTOR significantly enhanced, while inhibition of autophagy attenuated, PM-induced IL6 expression in HBE cells. Consistently, club-cell-specific deletion of Mtor aggravated, whereas loss of Atg5 in bronchial epithelium reduced, PM-induced airway inflammation. Interestingly, the augmented inflammatory responses caused by MTOR deficiency were markedly attenuated by blockage of downstream autophagy both in vitro and in vivo. Mechanistically, the dysregulation of MTOR-autophagy signaling was partially dependent on activation of upstream TSC2, and interacted with the TLR4-MYD88 to orchestrate the downstream NFKB activity and to regulate the production of inflammatory cytokines in airway epithelium. Moreover, inhibition of autophagy reduced the expression of EPS15 and the subsequent endocytosis of PM. Taken together, the present study provides a mechanistic explanation for how airway epithelium localized MTOR-autophagy axis regulates PM-induced airway injury, suggesting that activation of MTOR and/or suppression of autophagy in local airway might be effective therapeutic strategies for PM-related airway disorders. Abbreviations ACTB: actin beta; AKT: AKT serine/threonine kinase; ALI: air liquid interface; AP2: adaptor related protein complex 2; ATG: autophagy related; BALF: bronchoalveolar lavage fluid; COPD: chronic obstructive pulmonary disease; CXCL: C-X-C motif chemokine ligand; DOX: doxycycline; EGF: epidermal growth factor; EGFR: epidermal growth factor receptor; EPS15: epidermal growth factor receptor pathway substrate 15; HBE: human bronchial epithelial; H&E: hematoxylin & eosin; IKK: IKB kinase; IL: interleukin; LAMP2: lysosomal-associated membrane protein 2; LPS: lipopolysaccharide; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MTEC: mouse tracheal epithelial cells; MTOR: mechanistic target of rapamycin kinase; MYD88: MYD88 innate immune signal transduction adaptor; NFKB: nuclear factor of kappa B; NFKBIA: NFKB inhibitor alpha; PM: particulate matter; PtdIns3K: phosphatidylinositol 3-kinase; Rapa: rapamycin; RELA: RELA proto-oncogene, NFKB subunit; SCGB1A1: secretoglobin family 1A member 1; siRNA: small interfering RNAs; SQSTM1: sequestosome 1; TEM: transmission electronic microscopy; TLR4: toll like receptor 4; TSC2: TSC complex subunit 2.

Loxapine, an antipsychotic drug, suppresses intracellular multiple-antibiotic-resistant Salmonella enterica serovar Typhimurium in macrophages.

The emergence of multiple-antibiotic-resistant (MAR) Salmonella has been a serious threat worldwide. Salmonella can invade into host cells and evade the attacks of host humoral defenses and antibiotics. Thus, a new antibacterial agent capable of inhibiting intracellular Salmonella is highly needed. The anti-intracellular activity and cytotoxicity of drugs on intracellular bacteria and macrophages were assayed using intracellular CFU assay and MTT cell viability assay, respectively. The uptake of gentamicin into macrophage and the effect of autophagy inhibitor on loxapine's anti-intracellular Salmonella activity were assessed by using image-based high-content system. The expression of bacterial genes was measured by real-time PCR. The efflux pump activity of bacteria was measured by Hoechst accumulation assays. With our efforts, an antipsychotic drug, loxapine, was identified to exhibit high potency in suppressing intracellular MAR S. Typhimurium, Staphylococcus aureus, Shigella flexneri or Yersinia enterocolitica. Subsequent investigations indicated that loxapine's anti-intracellular bacteria activity was not associated with increased penetration of gentamicin into bacteria and macrophages. Loxapine didn't inhibit bacterial growth in broth at concentration up to 500μM and has no effect on Salmonella's type III secretion system genes' expression. Blockage of autophagy also didn't reverse loxapine's anti-intracellular activity. Lastly, loxapine suppressed bacterial efflux pump activity in all bacteria tested. Altogether, our data suggested that loxapine might suppress intracellular bacteria through inhibiting of bacterial efflux pumps. In light of its unique activity, loxapine represents a promising lead compound with translational potential for the development of a new antibacterial agent against intracellular bacteria.

Activation of Aurora A kinase increases YAP stability via blockage of autophagy

Transcription cofactor Yes-associated protein (YAP) plays an important role in cancer progression. Here, we found that Aurora A kinase expression was positively correlated with YAP in lung cancer. Aurora A depletion suppresses lung cancer cell colony formation, which could be reversed by YAP ectopic overexpression. In addition, activation of Aurora A increases YAP protein abundance through maintaining its protein stability. Consistently, the transcriptional activity of YAP is increased upon Aurora A activation. We further showed that shAURKA suppressed YAP expression in the absence of Lats1/2, indicating that Aurora A regulates YAP independently of Hippo pathway. Instead, Aurora A induced blockage of autophagy to up-regulate YAP expression. Collectively, our findings provide insights into regulatory mechanisms of YAP expression in lung cancer development.

Cancer-associated fibroblasts endow stem-like qualities to liver cancer cells by modulating autophagy.

PurposeBoth cancer-associated fibroblasts (CAFs) and liver cancer stem cells (LCSCs) play an important part in the tumorigenesis, development and metastasis of hepatocellular carcinoma (HCC). Moreover, the stem-like properties in HCC cells could be promoted by CAFs. However, the mechanism remains largely unknown.Patients and methodsWe used conditioned medium (CM) of CAFs to culture Huh7 cells. Stemness of the cells was then examined mainly by sphere formation assay while stemness-associated genes including Nanog, Sox2 and Oct4 were measured by Western blotting. Immunofluorescence staining, Transmission Electron Microscope as well as Western blotting were performed to detect the level of autophagy in Huh7 cells.ResultsIncreased level of stemness and autophagy was observed in HCC cells cultured in CAFs-CM compared to the control group. Activation of CAFs-induced autophagic flux could be inhibited by Chloroquine (CQ), which can accumulate LC3-II protein and increase punctate distribution of LC3 localization. Treatment of HCC cells with CQ effectively reversed the CAF-induced stemness, invasion, and metastasis ability in these cells. In vivo, Huh7 cells inoculated together with CAFs developed significantly larger tumors than Huh7 cells injected alone. Moreover, blockage of autophagy in Huh7 cells by CQ greatly reduced the growth of xenografted tumors of Huh7 cells combined with CAFs.ConclusionThese results reveal that CAFs are capable of promoting stemness and metastasis of HCC cells and blocking autophagy could markedly attenuate the stemness enhanced by CAFs, suggesting that targeting autophagy in HCC could be an effective strategy in HCC treatment.

P62/mTOR/LXRα pathway inhibits cholesterol efflux mediated by ABCA1 and ABCG1 during autophagy blockage

ObjectiveAtherosclerosis is a disease characterized by abnormal lipid metabolism, and the formation of foam cells is considered an early event of atherosclerosis. Intracellular cholesterol efflux mediated by ABCA1 and ABCG1 helps to reduce lipid accumulation in foam cells. Related studies have shown that autophagy and mTOR are involved in cholesterol efflux, but the role of p62, an autophagy substrate protein, has not been evaluated. MethodsTHP-1 derived macrophages were incubated with ox-LDL to establish a foam cell model and treated with different autophagy inducers. The effects of p62 on cholesterol efflux were investigated using overexpression vectors, gene silencing and western blotting. ResultsThis study showed a blockage of autophagy and decreased expression of ABCA1 and ABCG1 under the stress of excess ox-LDL in a concentration-dependent manner in THP-1 cells. Furthermore, the activation of autophagy led to increased expression of ABCA1 and ABCG1, as well as their upstream transcription factor LXRα, thereby promoting cholesterol efflux from foam cells. We also demonstrated that accumulated p62 played an important role during autophagy blockage, which was achieved by activating mTOR and then inhibited the expression of LXRα and its downstream target proteins ABCA1 and ABCG1. ConclusionIn conclusion, our experiments demonstrated that a p62/mTOR/LXRα signaling pathway was involved in cholesterol efflux mediated by ABCA1 and ABCG1 when autophagy blockage occurred. Our study offers a rationale for the development of autophagy and p62 as a new target for the treatment of atherosclerosis.

Final-2 targeted glycolysis mediated apoptosis and autophagy in human lung adenocarcinoma cells but failed to inhibit xenograft in nude mice

Natural products derived from fruits have multiple antitumor potential. However, very few have been developed for clinical therapy, due to the limited efficiency or insufficient study of their mechanism. Since lung cancer is the most common cancer in the world, there is still need to explore novel compounds but their molecular mechanisms remain elusive. In this study, a new compound Final-2 was synthesized. Final-2 exhibited antitumor activity in A549 cells by promoting apoptosis and blocking autophagy. Moreover, Final-2 significantly induced G0/G1 cycle arrest and inhibited cell malignancy. Intracellular molecular targets investigation showed that Final-2 inhibited the Gluts, which resulted in downregulation of glucose metabolism and the oncogene c-Myc and Kras expression in vitro. However, according the autophagy inhibitor CQ and Kras inhibitors test, low concentration of Final-2 showed some controversial effects. In A549 xenograft mice model, 10 mg/kg and 20 mg/kg of Final-2 showed no and partial tumor inhibition, respectively. Moreover, a high dose of Final-2 induced serious liver necrosis. Therefore, the results indicated that even though Final-2 was efficient in suppressing the cancer cell growth in vitro, it failed to inhibit tumors in vivo and showed significant liver toxicity, which was its limitation as a potential antitumor drug.

Quercetin and Sodium Butyrate Synergistically Increase Apoptosis in Rat C6 and Human T98G Glioblastoma Cells Through Inhibition of Autophagy.

This study investigated the efficacy of quercetin (QCT) in combination with sodium butyrate (NaB) in enhancing apoptosis in rat C6 and human T98G glioblastoma cells though blockage of autophagy under nutrient-starvation. The most synergistic doses of the drugs were determined to be 25µM QCT and 1mM NaB in both cell lines. After QCT and QCT + NaB treatments, autophagy quantification with acridine orange staining showed a drastic decrease in protective autophagy in the cells under nutrient-starvation. Decrease in autophagy was correlated with decreases in expression of Beclin-1 and LC3B II. Combination treatment increased the morphological signs of apoptosis including membrane blebbing, nuclear fragmentation, and chromatin condensation. Annexin V staining was also performed for detection and quantification of increases in apoptosis. Western blotting results showed that combination of QCT and NaB increased apoptosis by decreasing anti-apoptotic Bcl-2 and increasing pro-apoptotic Bax, decreasing survivin, activating caspase-3, and degrading poly (ADP-ribose) polymerase (PARP). This study demonstrated the therapeutic potentials of a novel combination therapy in inhibiting protective autophagy to enhance apoptosis in rat C6 and human T98G glioblastoma cells.

9za plays cytotoxic and proapoptotic roles and induces cytoprotective autophagy through the PDK1/Akt/mTOR axis in non-small-cell lung cancer.

Non-small-cell lung cancer (NSCLC) is an aggressive subtype of pulmonary carcinomas with high mortality. However, chemotherapy drug resistance and high recurrence rates hinder the curative effect of platinum-based first-line chemotherapy, which makes it urgent to develop new antitumor drugs for NSCLC. 9za, a new candidate drug synthesized by our research group, has been verified with potent antilung cancer activity in preliminary experiments. However, the underlying molecular mechanism of 9za remains largely vague. This work revealed that 9za could play important cytotoxic and proapoptotic roles in NSCLC cells. Moreover, 9za could induce autophagy and promote autophagy flux. Interestingly, the cytotoxic and proapoptotic roles were significantly dependent on 9za-induced cytoprotective autophagy. That is, the coadministration of 9za with an autophagy inhibitor such as chloroquine or 3-methyladenine exhibited increased cytotoxic and proapoptotic effects compared with 9za treatment alone. In addition, 9za exposure suppressed the phosphorylation of phosphoinositide-dependent protein kinase 1 (PDK1), protein kinase B (Akt), mammalian targets of rapamycin (mTOR), p70 S6 kinase, and 4E binding protein 1 by a dose-dependent way, manifesting that the Akt/mTOR axis was implicated in 9za-induced autophagy. In addition, the overexpression of PDK1 resulted in increased phosphorylation of PDK1 and Akt and blocking of 9za-mediated autophagy. These data showed that the PDK1/Akt/mTOR pathway was involved in 9za-induced autophagy. Hence, this work provides a theoretical basis for exploiting 9za as a new antilung cancer candidate drug and hints that the combination of 9za with an autophagy inhibitor is a feasible alternative approach for the therapy of NSCLC.

Sirt6-induced autophagy restricted TREM-1-mediated pyroptosis in ox-LDL-treated endothelial cells: relevance to prognostication of patients with acute myocardial infarction

Inflammation mediated by myeloid cells trigger receptors 1 (TREM-1) is important for atherosclerosis development, while sirtuin 6 (Sirt6) levels decrease in atheroscleoritc plaque. Here we demonstrate that oxidatively modified low density lipoprotein (ox-LDL)-treated endothelial cells (ECs) exhibited increased TREM-1-mediated pyroptosis and decreased Sirt6-induced autophagy. We show that high sTREM-1 and low sSirt6 levels were independent predictors of boosted endothelial microparticles (EMPs) on admission, and were associated with increased risk for all-cause mortality and major adverse cardiovascular events (MACE) at median 24 months (interquartile range, 18–26) follow-up in acute myocardial infarction (AMI) patients. Additionally, blockage of Sirt6-induced autophagy led to augmented TREM-1-mediated pyroptosis, whereas Sirt6 overexpression attenuated ECs inflammation and pyroptosis following ox-LDL treatment. Our findings indicate that TREM-1 and in a reversed trend Sirt6 appeared to be markers of endothelial inflammation with potential for use in risk stratification.

Hsp90 Inhibitor SNX-2112 Enhances TRAIL-Induced Apoptosis of Human Cervical Cancer Cells via the ROS-Mediated JNK-p53-Autophagy-DR5 Pathway.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent cancer cell apoptosis-inducing factor that can induce apoptosis in a variety of cancer cells. However, resistance to TRAIL in cancer cells is a huge obstacle in creating effective TRAIL-targeted clinical therapies. Thus, agents that can either enhance the effect of TRAIL or overcome its resistance are needed. In this study, we combined TRAIL with SNX-2112, an Hsp90 inhibitor we previously developed, to explore the effect and mechanism that SNX-2112 enhanced TRAIL-induced apoptosis in cervical cancer cells. Our results showed that SNX-2112 markedly enhanced TRAIL-induced cytotoxicity in HeLa cells, and this combination was found to be synergistic. Additionally, we found that SNX-2112 sensitized TRAIL-mediated apoptosis caspase-dependently in TRAIL-resistant HeLa cells. Mechanismly, SNX-2112 downregulated antiapoptosis proteins, including Bcl-2, Bcl-XL, and FLIP, promoted the accumulation of reactive oxygen species (ROS), and increased the expression levels of p-JNK and p53. ROS scavenger NAC rescued SNX-2112/TRAIL-induced apoptosis and suppressed SNX-2112-induced p-JNK and p53. Moreover, SNX-2112 induced the upregulation of death-receptor DR5 in HeLa cells. The silencing of DR5 by siRNA significantly decreased cell apoptosis by the combined effect of SNX-2112 and TRAIL. In addition, SNX-2112 inhibited the Akt/mTOR signaling pathway and induced autophagy in HeLa cells. The blockage of autophagy by bafilomycin A1 or Atg7 siRNA abolished SNX-2112-induced upregulation of DR5. Meanwhile, ROS scavenger NAC, JNK inhibitor SP600125, and p53 inhibitor PFTα were used to verify that autophagy-mediated upregulation of DR5 was regulated by the SNX-2112-stimulated activation of the ROS-JNK-p53 signaling pathway. Thus, the combination of SNX-2112 and TRAIL may provide a novel strategy for the treatment of human cervical cancer by overcoming cellular mechanisms of apoptosis resistance.

Lysosomal Pathways and Autophagy Distinctively Control Endothelial Cell Behavior to Affect Tumor Vasculature

Cancer cell-stromal cell crosstalk is orchestrated by a plethora of ligand-receptor interactions generating a tumor microenvironment (TME) which favors tumor growth. The high pro-angiogenic nature of the TME perpetuates the chaotic network of structurally immature, low pericyte-covered vessels characteristic of the tumor vasculature. We previously demonstrated that chloroquine (CQ) -a lysosomotropic agent used as first-generation autophagy blocker in clinical trials- induced tumor vessel normalization and reduced tumor hypoxia. CQ improved both vessel structure and maturation, whereas the conditional knockout of the crucial autophagy gene Atg5 in endothelial cells (ECs) did not, thus highlighting a potential differential role for EC-associated autophagy and the lysosomes in pathological tumor angiogenesis. However, how CQ or ATG5-deficiency in ECs affect angiogenic signals regulating EC-pericyte interface and therefore vessel maturation, remains unknown. Here, we show that in ECs CQ constrained VEGF-A-mediated VEGF receptor (VEGFR)2 phosphorylation, a driver of angiogenic signaling. In the presence of CQ we observed increased expression of the decoy receptor VEGFR1 and of a lower molecular weight form of VEGFR2, suggesting receptor cleavage. Consequently, VEGF-A-driven EC spheroid sprouting was reduced by CQ treatment. Furthermore, CQ significantly affected the transcription and secretion of platelet-derived growth factor (PDGF)-AB/BB (upregulated) and Endothelin-1 (EDN1, downregulated), both modulators of perivascular cell (PC) behavior. In contrast, silencing of ATG5 in ECs had no effect on VEGFR2 to VEGFR1 ratio nor on PDGFB and EDN1 expression. Accordingly, mice harboring B16F10 melanoma tumors treated with CQ, displayed both an increased number of αSMA+ PCs covering tumor vessels and co-expressed PDGF receptor-β, enabling PDGF ligand dependent recruitment. Moreover, upon CQ treatment the tumoral expression of angiopoietin-1 (Angpt1), which retains mural cells, and induces vessel stabilization by binding to the EC-localized cognate receptor (TIE2), was increased thus supporting the vessel normalization function of CQ. These features associated with improved tumor vasculature were not phenocopied by the specific deletion of Atg5 in ECs. In conclusion, this study further unravels endothelial cell autonomous and non-autonomous mechanisms by which CQ “normalizes” the intercellular communication in the tumor vasculature independent of autophagy.

\u03b2-Thujaplicin induces autophagic cell death, apoptosis, and cell cycle arrest through ROS-mediated Akt and p38/ERK MAPK signaling in human hepatocellular carcinoma

Hepatocellular carcinoma (HCC), a common liver malignancy worldwide, has high morbidity and mortality. β-Thujaplicin, a tropolone derivative, has been used in some health-care products and clinical adjuvant drugs, but its use for HCC is unknown. In this study, we found that β-Thujaplicin inhibits the growth of HCC cells, but not normal liver cells, with nanomolar potency. Mechanistically, we found that β-Thujaplicin could induce autophagy, as judged by western blot, confocal microscopy, and transmission electron microscopy. Further using β-Thujaplicin combined with an autophagy blocker or agonist treatment HepG2 cells, we found that β-Thujaplicin induced autophagic cell death (ACD) mediated by ROS caused inhibition of the Akt-mTOR signaling pathway. Moreover, β-Thujaplicin triggered HepG2 apoptosis and increased cleaved PARP1, cleaved caspase-3, and Bax/Bcl-2 ratio, which indicated that β-Thujaplicin induced apoptosis mediated by the mitochondrial-dependent pathway. We also found that increased expression of p21 and decreased expression of CDK7, Cyclin D1, and Cyclin A2 participating in β-Thujaplicin caused the S-phase arrest. It seems that β-Thujaplicin exerts these functions by ROS-mediated p38/ERK MAPK but not by JNK signaling pathway activation. Consistent with in vitro findings, our in vivo study verified that β-Thujaplicin treatment significantly reduced HepG2 tumor xenograft growth. Taken together these findings suggest that β-Thujaplicin have an ability of anti-HCC cells and may conducively promote the development of novel anti-cancer agents.

Synthesis and evaluation of novel benzotropolones as Atg4B inhibiting autophagy blockers

Autophagy is an intracellular degradation/recycling pathway that provides nutrients and building blocks to cellular metabolism and keeps the cytoplasm clear of obsolete proteins and organelles. During recent years, dysregulated autophagy activity has been reported to be a characteristic of many different disease types, including cancer and neurodegenerative disorders. This has created a strong case for development of autophagy modulating compounds as potential treatments for these diseases. Inhibitors of autophagy have been proposed as a therapeutic intervention in, e.g., advanced cancer, and inhibiting the cysteine protease Atg4B has been put forward as a main strategy to block autophagy. We recently identified and demonstrated -both in vitro and in vivo - that compounds with a benzotropolone basic structure targeting Atg4B, can significantly slow down tumor growth and potentiate the effect of classical chemotherapy. In this study we report the synthesis and inhibition profile of new benzotropolone derivatives with additional structural modifications at 6 different positions. To obtain a solid inhibition profile, all compounds were evaluated on three levels, including two cell-based assays to confirm autophagy and intracellular Atg4B inhibition and an SDS-PAGE-based experiment to assess in vitro Atg4B affinity. Several molecules with a promising profile were identified.

Blocking autophagy flux promotes interferon-alpha-mediated apoptosis in head and neck squamous cell carcinoma.

Despite multiple antitumor activities, interferon-alpha (IFNα) therapy alone is less effective in solid tumors. Autophagy has been reported to play a key role in tumor chemoresistance. Therefore, it is meaningful to explore whether autophagy can be activated by IFNα in head and neck squamous cell carcinoma (HNSCC) and serve as a potential target to improve efficacy of IFNα therapy. In this study, we report that IFNα not only exhibits anti-proliferation activity and induces apoptosis, but also activates autophagy in HNSCC cells. Moreover, silencing autophagy-related protein 5 (ATG5) and signal transducer and activator of transcription 1 (STAT1) suppresses autophagy flux. Furthermore, IFNα and autophagy inhibitors (hydroxychloroquine and wortmannin) show clear synergistic effects on inhibiting growth and promoting apoptosis in HNSCC cells and xenograft models. Our findings indicate that IFNα-induced autophagy plays a cytoprotective role and blocking autophagy flux promotes IFNα-mediated apoptosis in HNSCC. These results suggest that the combination of IFNα and autophagy inhibitors represents a novel strategy for HNSCC treatment.