Autophagy-mediated Cell Death Research Articles

Novel smac mimetic APG-1387 elicits ovarian cancer cell killing through TNF-alpha, Ripoptosome and autophagy mediated cell death pathway

BackgroundOvarian cancer is a deadly disease. Inhibitors of apoptosis proteins (IAPs) are key regulators of apoptosis and are frequently dysregulated in ovarian cancer. Overexpression of IAPs proteins has been correlated with tumorigenesis, treatment resistance and poor prognosis. Reinstalling functional cell death machinery by pharmacological inhibition of IAPs proteins may represent an attractive therapeutic strategy for treatment of ovarian cancer.MethodsCCK-8 and colony formation assay was performed to examine cytotoxic activity. Apoptosis was analyzed by fluorescence microscopy, flow cytometry and TUNEL assay. Elisa assay was used to determine TNFα protein. Caspase activity assay was used for caspase activation evaluation. Immunoprecipitation and siRNA interference were carried out for functional analysis. Western blotting analysis were carried out to test protein expression. Ovarian cancer cell xenograft nude mice model was used for in vivo efficacy evaluation.ResultsAPG-1387 demonstrated potent inhibitory effect on ovarian cancer cell growth and clonogenic cell survival. APG-1387 induced RIP1- and TNFα-dependent apoptotic cell death in ovarian cancer through downregulation of IAPs proteins and induction of caspase-8/FADD/RIP1 complex, which drives caspase-8 activation. NF-κB signaling pathway was activated upon APG-1387 treatment and RIP1 contributed to NF-κB activation. APG-1387 induced cytoprotective autophagy while triggering apoptosis in ovarian cancer cells and inhibition of autophagy enhanced APG-1387-induced apoptotic cell death. APG-1387 exhibited potent antitumor activity against established human ovarian cancer xenografts.ConclusionsOur results demonstrate that APG-1387 targets IAPs proteins to potently elicit apoptotic cell death in vitro and in vivo, and provide mechanistic and applicable rationale for future clinical evaluation of APG-1387 in ovarian cancer.

Long noncoding RNA RP4 functions as a competing endogenous RNA through miR-7-5p sponge activity in colorectal cancer

AIMTo investigate the role of long noncoding RNA (lncRNA) RP4 in colorectal cancer.METHODSLentivirus-mediated lncRNA RP4 overexpression and knockdown were performed in the colorectal cancer cell line SW480. Cell proliferation, tumor growth, and early apoptosis were evaluated by a cell counting kit-8 assay, an in vivo xenograft tumor model, and annexin V/propidium iodide staining, respectively. Analysis of the lncRNA RP4 mechanism involved assessment of the association of its expression with miR-7-5p and the SH3GLB1 gene. Western blot analysis was also performed to assess the effect of lncRNA RP4 on the autophagy-mediated cell death pathway and phosphatidylinositol-3-kinase (PI3K)/Akt signaling.RESULTSCell proliferation, tumor growth, and early apoptosis in SW480 cells were negatively regulated by lncRNA RP4. Functional experiments indicated that lncRNA RP4 directly upregulated SH3GLB1 expression by acting as a competing endogenous RNA (ceRNA) for miR-7-5p. This interaction led to activation of the autophagy-mediated cell death pathway and de-repression of PI3K and Akt phosphorylation in colorectal cancer cells in vivo.CONCLUSIONOur results demonstrated that lncRNA RP4 is a ceRNA that plays an important role in the pathogenesis of colorectal cancer, and could be a potential therapeutic target for colorectal cancer treatment.

PM2.5 induces autophagy-mediated cell death via NOS2 signaling in human bronchial epithelium cells.

The biggest victim of ambient air pollution is the respiratory system. Mainly because of the harmful components, especially the particulate matters with an aerodynamic diameter of ≤ 2.5µm (PM2.5), can be directly inhaled and deeply penetrate into the lung alveoli, thus causing severe lung dysfunction, including chronic cough, bronchitis and asthma, even lung cancer. Unfortunately, the toxicological mechanisms of PM2.5 associations with these adverse respiratory outcomes have still not been clearly unveiled. Here, we found that PM2.5 rapidly induced inflammatory responses, oxidative injure and cell death in human bronchial epithelium cells through upregulation of IL-6 expression, ROS production and apoptosis. Furthermore, PM2.5 specifically induced nitric oxide synthase 2 (NOS2) expression and NO generation to elevate excessive autophagy. Finally, disruption of NOS2 signaling effectively blocked autophayosome formation and the subsequent cell death. Our novel findings systemically reveled the role of autophagy-mediated cell death in PM2.5-treated human bronchial epithelium cells and provided potential strategy for future clinic intervention.

Sinomenine Hydrochloride Inhibits Human Glioblastoma Cell Growth through Reactive Oxygen Species Generation and Autophagy-Lysosome Pathway Activation: An In Vitro and In Vivo Study.

Glioblastoma is the most common malignant primary brain tumor, and it is one of the causes of cancer fatality in both adult and pediatric populations. Patients with glioblastoma require chemotherapy after surgical resection and radiotherapy. Therefore, chemotherapy constitutes a viable approach for the eradication of glioblastoma cells. In this study, the anti-tumor activity of sinomenine hydrochloride (SH) was evaluated in U87 and SF767 cells in vitro and in vivo. The results showed that SH potently inhibited U87 and SF767 cell viability and did not cause caspase-dependent cell death, as demonstrated by the absence of significant early apoptosis and caspase-3 cleavage. Instead, SH activated an autophagy-mediated cell death pathway, as indicated by the accumulated microtubule-associated protein light chain 3B (LC3B)-II, triggered autophagic flux and enhanced cell viability after pretreatment with autophagy inhibitors. SH-mediated autophagy in the two cell lines was implicated in reactive oxygen species (ROS) generation, protein kinase B (Akt)-mammalian target of rapamycin (mTOR) pathway suppression and c-Jun NH2-terminal kinase (JNK) pathway activation. The ROS antioxidant N-acetylcysteine (NAC), the Akt-specific activator insulin-like growth factor-1 (IGF-1) and the JNK-specific inhibitor SP600125 attenuated SH-induced autophagy. Moreover, ROS activated autophagy via the Akt-mTOR and JNK pathways. Additionally, SH treatment may promote lysosome biogenesis through activating transcription factor EB (TFEB). The in vivo study found that SH effectively suppressed glioblastoma growth without exhibiting significant toxicity. In conclusion, our findings reveal a novel mechanism of action of SH in cancer cells via the induction of autophagy through ROS generation and autophagy-lysosome pathway activation; these findings also supply a new potential therapeutic agent for the treatment of human glioblastoma.

Molecular Elucidation of Biological Response to Mesoporous Silica Nanoparticles in Vitro and in Vivo.

Biomedical applications of mesoporous silica nanoparticles (MSNs) require efficient cellular uptake and low toxicity. The purpose of this study is to investigate the cellular uptake and toxicity of MSNs with different sizes and charges (50, 100, and 250 nm with a positive surface charge and 100 nm with a negative surface charge) exposed to human monocyte-derived macrophages, lung epithelium BEAS-2B cells, and mice using genome-wide gene expression analysis and cellular/animal-level end point tests. We found that MSNs can be taken up into cells through endocytosis in a charge- and size-dependent manner, with positively charged and larger MSNs being more easily taken up into the cells by recruiting more types of endocytotic pathways for more cellular uptake. Moreover, the cytotoxicity of MSNs could be correlated with the amount of MSNs taken up by cells, which positively correlates to the particle size and dosage. Therefore, only positively charged and larger MSNs (≥100 nm) during higher treatment doses (≥500 μg mL-1) resulted in a sufficient accumulation of internalized MSNs in cells to induce significant release of reactive oxygen species (ROS) and oxidative stress, inflammatory gene upregulation through NF-κB and AP-1, and eventually autophagy-mediated necrotic cell death. Furthermore, genome-wide gene expression analysis could reflect the above in vitro cellular damages and corresponding in vivo injuries in mice, indicating that specific gene expression footprints may be used for assessing the safety of nanoparticles. The present finding provides some insights into the rational design of effective MSN-based drug/gene delivery systems and biomedical applications.

Inhibition of CK2 by CX4945 to induce autophagy-mediated cell death through dephosphorylating acetyl-CoA carboxylase in squamous cell carcinoma of head and neck cancer.

e17503 Background: Accumulating evidence has shown that overexpression of casein kinase 2 (CK2) in patients with squamous cell carcinoma of head and neck (SCCHN) correlates with worsened survival. Strong expression of phosphorylated acetyl CoA carboxylase (pACC) was found to be an independent prognostic marker for patients with node-positive SCCHN. Computational analysis showed that ACC was a potential substrate for CK2, and CK2 inhibition can suppress ACC phosphorylation in non-cancer cells. CX-4945, also known as silmitasertib, is an orally administered, highly specific, ATP-competitive inhibitor of CK2 and is under clinical investigation for treating malignancies. Methods: We investigated the anticancer activity of CX-4945 in 3 aggressive SCCHN cell lines. Autophagy induction and interference with cellular metabolism were characterized. Results: CX-4945 induced autophagic cell death in SCCHN cells. CX-4945 treatment caused time- and dose-dependent lipid droplet accumulation, inhibition of ACC phosphorylation, depressed cellular levels of acetyl Co-A, and severely suppressed mitochondrial respiration, without altering the expression of stress-related proteins in the endoplasmic reticulum. Conclusions: The findings demonstrated that inhibition of CK2 by CX-4945 contributed to deranged cellular energy metabolism in SCCHN cells, leading to autophagy-mediated programmed cell death. CX-4945 targeting to CK2 may be a promising therapeutic strategy against SCCHN cancer.

Zinc oxide nanoparticle energy band gap reduction triggers the oxidative stress resulting into autophagy-mediated apoptotic cell death

The physico-chemical properties of nanoparticle (NP), such as particle size, surface defects, crystallinity and accessible surface, affect NP photocatalytic activity that in turn defines the NP cytotoxic propensity. Since zinc oxide nanoparticle (ZnONP) energy band gap falls in a range of a semiconductor, the particle possesses photocatalytic activity. Hence, the study correlates energy band gap with cytotoxic propensity of ZnONP. To this end, ZnONPs with varying energy band gap are fabricated by varying calcination temperature. Cytotoxic propensity of the fabricated ZnONPs against HT1080 cell indicates that the particle with least energy band gap shows highest cytotoxicity. The data also indicate that the cytotoxicity is triggered primarily through reactive oxygen species (ROS)-mediated pathway. Additionally, the comet assay and γH2AX activity assay reveal that decreasing energy band gap of the particle increases DNA damaging propensity. Furthermore, cell cycle analysis indicates that the cell treatment with decreasing energy band gap ZnONP results in significant increase in cell population fraction in subG1 phase. Whereas, acridine orange binding assay and increased expression level of LC3II indicate that the cell tries to recover the stress by scavenging damaged cellular biomolecules and ROS using autophagosomes. Nevertheless, cell with the non-recoverable damages led into apoptotic cell death, as confirmed by Annexin V apoptosis assay, DNA fragmentation assay and 4,6-Diamidino-2-phenylindole dihydrochloride (DAPI) staining.

3-Decylcatechol induces autophagy-mediated cell death through the IRE1α/JNK/p62 in hepatocellular carcinoma cells.

The natural, phenolic lipid urushiol exhibits both antioxidant and anticancer activities; however, its biological activity on hepatocellular carcinoma (HCC) has not been previously investigated. Here, we demonstrate that an urushiol derivative, 3-decylcatechol (DC), induces human HCC Huh7 cell death by induction of autophagy. DC initiates the autophagic process by activation of the mammalian target of rapamycin signaling pathway via Unc-51-like autophagy activating kinase 1, leading to autophagosome formation. The autophagy inhibitor, chloroquine, suppressed autolysosome formation and cell death induction by DC, indicating an autophagic cell death. Interestingly, DC also activated the endoplasmic reticulum (ER) stress response that promotes autophagy via p62 transcriptional activation involving the inositol-requiring enzyme 1α/c-Jun N-terminal kinase/c-jun pathway. We also show that cytosolic calcium mobilization is necessary for the ER stress response and autophagy induction by DC. These findings reveal a novel mechanism by which this urushiol derivative induces autophagic cell death in HCC.

Attenuation of γ‐secretase mediated activation of Notch signaling induces autophagic cell death in triple negative breast cancer cells

Notch signaling is reported to be deregulated in several malignancies including breast. γ‐secretase, a multi‐subunit enzyme complex, catalyzes the cleavage of Notch receptor to release the Notch intracellular domain (NICD) in the cytosol, which then translocates into nucleus to activate the downstream signaling. Hence pharmacological inhibition of γ‐secretase might lead to the subsequent inhibition of Notch signaling in cancer cells.In search of novel γ‐secretase inhibitors (GSIs), we screened a series of triazole‐based compounds, for their potential to bind γ‐secretase and we observed that 3‐(3′4′,5′‐Trimethoxyphenyl)‐5‐(N‐methyl‐3′‐indolyl)‐1,2,4‐triazole compound (also known as NMK‐T‐057) can bind to γ‐secretase complex. Very interestingly, NMK‐T‐057 was found to inhibit proleiferation, colony forming ability, motility in various triple negative breast cancer cells such as MDA‐MB‐231, MDA‐MB‐468, 4T1 and also MCF‐7, with a negligible cytotoxicity against non‐cancerous MCF‐10A, PBMC and WI38. Moreover NMK‐T‐057 treatment resulted in the reprogramming of the EMT process in TNBCs, accompanied by the drastic reduction of CD44high/ CD24low population. NMK‐T‐057 also inhibited the spheroid forming ability of TNBCs and reduced the stem cell enriched side population. It was also observed that in NMK‐T‐057 treated cells, Notch‐1 and its downstream targets such as Hes‐1, pAkt, and Cyclin D1 were significantly down regulated. The in silico study revealing the affinity of NMK‐T‐057 towards γ‐secretase, was further validated by fluorescence based γ‐secretase activity assay, which confirmed the inhibition of γ‐secretase activity in NMK‐T‐057 treated TNBC cells.Autophagy plays a dual role in cancer depending on its role in the survival or death of cancer cells. The relationship between autophagy and Notch signaling is not clear and only a few reports have come across in highly reputed journals in last one year. We observed that NMK‐T‐057 induces significant autophagic responses in TNBCs and administration of the autophagy inhibitor 3‐MA, attenuates NMK‐T‐057 induced cell death. Similar results were observed on the treatment of TNBC cells with commercially available γ‐secretase inhibitor DAPT. Thus we might conclude that NMK‐T‐057 could be potential drug candidate against breast cancer, which can trigger autophagy mediated cell death in breast cancer cells by inhibiting the γ‐secretase – mediated activation of Notch‐signaling.Support or Funding InformationThis work is supported by Dr. D. S. Korhari Postdoctoral Fellowship Scheme, UGC, Govt. of India.

HDAC4 and HDAC6 sustain DNA double strand break repair and stem-like phenotype by promoting radioresistance in glioblastoma cells

The role of histone deacetylase (HDAC) 4 and 6 in glioblastoma (GBM) radioresistance was investigated. We found that tumor samples from 31 GBM patients, who underwent temozolomide and radiotherapy combined treatment, showed HDAC4 and HDAC6 expression in 93.5% and 96.7% of cases, respectively. Retrospective clinical data analysis demonstrated that high-intensity HDAC4 and/or HDAC6 immunostaining was predictive of poor clinical outcome. In vitro experiments revealed that short hairpin RNA-mediated silencing of HDAC4 or HDAC6 radiosensitized U87MG and U251MG GBM cell lines by promoting DNA double-strand break (DSBs) accumulation and by affecting DSBs repair molecular machinery. We found that HDAC6 knock-down predisposes to radiation therapy-induced U251MG apoptosis- and U87MG autophagy-mediated cell death. HDAC4 silencing promoted radiation therapy-induced senescence, independently by the cellular context. Finally, we showed that p53WT expression contributed to the radiotherapy lethal effects and that HDAC4 or HDAC6 sustained GBM stem-like radioresistant phenotype. Altogether, these observations suggest that HDAC4 and HDAC6 are guardians of irradiation-induced DNA damages and stemness, thus promoting radioresistance, and may represent potential prognostic markers and therapeutic targets in GBM.

Dihydroceramide accumulation mediates cytotoxic autophagy of cancer cells via autolysosome destabilization

ABSTRACTAutophagy is considered primarily a cell survival process, although it can also lead to cell death. However, the factors that dictate the shift between these 2 opposite outcomes remain largely unknown. In this work, we used Δ9-tetrahydrocannabinol (THC, the main active component of marijuana, a compound that triggers autophagy-mediated cancer cell death) and nutrient deprivation (an autophagic stimulus that triggers cytoprotective autophagy) to investigate the precise molecular mechanisms responsible for the activation of cytotoxic autophagy in cancer cells. By using a wide array of experimental approaches we show that THC (but not nutrient deprivation) increases the dihydroceramide:ceramide ratio in the endoplasmic reticulum of glioma cells, and this alteration is directed to autophagosomes and autolysosomes to promote lysosomal membrane permeabilization, cathepsin release and the subsequent activation of apoptotic cell death. These findings pave the way to clarify the regulatory mechanisms that determine the selective activation of autophagy-mediated cancer cell death.

Cinobufagin induces autophagy-mediated cell death in human osteosarcoma U2OS cells through the ROS/JNK/p38 signaling pathway

The main objective of this study was to explore whether autophagy could be triggered by cinobufagin, and to clarify the role of autophagy in the antitumor effects of cinobufagin on U2OS cells and the underlying mechanisms. U2OS cells were exposed to 15, 30, 60 and 120 mg/l cinobufagin for 0, 12, 24 and 48 h. An MTT assay was used to measure cell viability. FITC-Annexin V/PI staining and flow cytometry were used to analyze the apoptotic ratio, while apoptotic morphological changes were assessed by PI and Hoechst 33258 viable cell staining. The effects of autophagy on the cells were investigated with GFP-LC3b green fluorescence plasmid transfection and transmission electron microscopy. The levels of caspase-3, -8, - 9, cleaved PARP, LC3-II/LC3-I, p62 and the activation of JNK/p-38 were detected by western blot analysis. Reactive oxygen species (ROS) fluorescence intensity was examined under fluorescence microscopy with an analysis software system. Cell proliferation was obviously inhibited by cinobufagin in a dose- and time-dependent manner. The apoptosis ratio was gradually increased with treatment time as evidenced by flow cytometric analysis and Hoechst 33258 staining. Exposure to cinobufagin resulted in the activation of caspase-3, -8, -9, as well as cleaved PARP which indicated that cinobufagin induced caspase-dependent apoptosis. Autophagy was confirmed in the cinobufagin-treated cells as evidenced by formation of autophagosomes, accumulation of GFP-LC3 fluorescence particles as well as the upregulation of LC3-II/LC3-I levels. Inhibition of autophagy diminished apoptosis as detected by the MTT assays. Moreover the percentage of apoptotic cells decreased following pretreatment with 3-MA, CQ and si-beclin-1. Cinobufagin also induced phosphorylation of the JNK and p38 signaling pathway as well as ROS generation. The JNK and p38 inhibitors significantly attenuated coexistence of apoptosis and autophagy-related proteins. The ROS scavenger also prevented phosphorylation of the JNK and p38 signaling pathway. Our research proved that cinobufagin triggered apoptosis and autophagic cell death via activation of the ROS/JNK/p-38 axis.

Targeting FXYD2 by cardiac glycosides potently blocks tumor growth in ovarian clear cell carcinoma.

Ovarian clear cell carcinoma (OCCC) is an aggressive neoplasm with a high recurrence rate that frequently develops resistance to platinum-based chemotherapy. There are few prognostic biomarkers or targeted therapies exist for patients with OCCC. Here, we identified that FXYD2, the modulating subunit of Na+/K+-ATPases, was highly and specifically expressed in clinical OCCC tissues. The expression levels of FXYD2 were significantly higher in advanced-stage of OCCC and positively correlated with patients' prognoses. Silencing of FXYD2 expression in OCCC cells inhibited Na+/K+-ATPase enzyme activity and suppressed tumor growth via induction of autophagy-mediated cell death. We found that high FXYD2 expression in OCCC was transcriptionally regulated by the transcriptional factor HNF1B. Furthermore, up-regulation of FXYD2 expression significantly increased the sensitivity of OCCC cells to cardiac glycosides, the Na+/K+-ATPase inhibitors. Two cardiac glycosides, digoxin and digitoxin, had a great therapeutic efficacy in OCCC cells in vitro and in vivo. Taken together, our results demonstrate that FXYD2 is functionally upregulated in OCCC and may serve as a promising prognostic biomarker and therapeutic target of cardiac glycosides in OCCC.

N-Acetylcysteine in Combination with IGF-1 Enhances Neuroprotection against Proteasome Dysfunction-Induced Neurotoxicity in SH-SY5Y Cells

Ubiquitin proteasome system (UPS) dysfunction has been implicated in the development of many neuronal disorders, including Parkinson's disease (PD). Previous studies focused on individual neuroprotective agents and their respective abilities to prevent neurotoxicity following a variety of toxic insults. However, the effects of the antioxidant N-acetylcysteine (NAC) on proteasome impairment-induced apoptosis have not been well characterized in human neuronal cells. The aim of this study was to determine whether cotreatment of NAC and insulin-like growth factor-1 (IGF-1) efficiently protected against proteasome inhibitor-induced cytotoxicity in SH-SY5Y cells. Our results demonstrate that the proteasome inhibitor, MG132, initiates poly(ADP-ribose) polymerase (PARP) cleavage, caspase 3 activation, and nuclear condensation and fragmentation. In addition, MG132 treatment leads to endoplasmic reticulum (ER) stress and autophagy-mediated cell death. All of these events can be attenuated without obvious reduction of MG132 induced protein ubiquitination by first treating the cells with NAC and IGF-1 separately or simultaneously prior to exposure to MG132. Moreover, our data demonstrated that the combination of the two proved to be significantly more effective for neuronal protection. Therefore, we conclude that the simultaneous use of growth/neurotrophic factors and a free radical scavenger may increase overall protection against UPS dysfunction-mediated cytotoxicity and neurodegeneration.

Time depended Bcl-2 inhibition might be useful for a targeted drug therapy.

BackgroundOver expression of Bcl-2 is frequently observed in several types of cancers and it is one of the prognostic markers in breast cancer. The importance of the Bcl-2 protein as ideal therapeutic target is the dual role of inhibiting apoptosis and autophagy-mediated cell death. Thus, the bcl-2 targeting may be a strategy of choice to improve treatment efficacy and overcome drug resistance to cancer chemotherapy. For this reason, we designed the siRNA mediated silencing of the Bcl-2 gene in the MCF-7 breast cancer cell line.ObjectivesThe purpose of this research was to investigate the effective Bcl-2 gene silencing by our homemade siRNA, more than previous study. Our data demonstrated that specific inhibition of the Bcl-2 by siRNA induces approximately more than 90 % gene silencing.MethodsMCF-7 Cell lines were treated by homemade Bcl-2siRNA for the first time and control siRNA that was transfected with nanoparticle. The cells harvested at 24, 48 and 72 h and transcription level of Bcl-2 was examined by Real Time -PCR analysis. The drug sensitivity was detected by using LDH assay test. Finally Anexin V-FITC test was performed for evaluation of apoptosis.ResultsIn the present study, results showed that targeting the specific sequence of the Bcl-2 by our homemade siRNA in the MCF7 cell line and its effect was more obvious in 24 h in contrast to 48 and 72 h.ConclusionsHowever, we showed here a time dependent blocking of the bcl-2 transcript that might lead to cell dead due autophagy, and not necessarily to apoptosis.

Andrographolide Analogue Induces Apoptosis and Autophagy Mediated Cell Death in U937 Cells by Inhibition of PI3K/Akt/mTOR Pathway.

BackgroundCurrent chemotherapeutic agents based on apoptosis induction are lacking in desired efficacy. Therefore, there is continuous effort to bring about new dimension in control and gradual eradication of cancer by means of ever evolving therapeutic strategies. Various forms of PCD are being increasingly implicated in anti-cancer therapy and the complex interplay among them is vital for the ultimate fate of proliferating cells. We elaborated and illustrated the underlying mechanism of the most potent Andrographolide analogue (AG–4) mediated action that involved the induction of dual modes of cell death—apoptosis and autophagy in human leukemic U937 cells.Principal FindingsAG–4 induced cytotoxicity was associated with redox imbalance and apoptosis which involved mitochondrial depolarisation, altered apoptotic protein expressions, activation of the caspase cascade leading to cell cycle arrest. Incubation with caspase inhibitor Z-VAD-fmk or Bax siRNA decreased cytotoxic efficacy of AG–4 emphasising critical roles of caspase and Bax. In addition, AG–4 induced autophagy as evident from LC3-II accumulation, increased Atg protein expressions and autophagosome formation. Pre-treatment with 3-MA or Atg 5 siRNA suppressed the cytotoxic effect of AG–4 implying the pro-death role of autophagy. Furthermore, incubation with Z-VAD-fmk or Bax siRNA subdued AG–4 induced autophagy and pre-treatment with 3-MA or Atg 5 siRNA curbed AG–4 induced apoptosis—implying that apoptosis and autophagy acted as partners in the context of AG–4 mediated action. AG–4 also inhibited PI3K/Akt/mTOR pathway. Inhibition of mTOR or Akt augmented AG–4 induced apoptosis and autophagy signifying its crucial role in its mechanism of action.ConclusionsThus, these findings prove the dual ability of AG–4 to induce apoptosis and autophagy which provide a new perspective to it as a potential molecule targeting PCD for future cancer therapeutics.

BC3EE2,9B, a synthetic carbazole derivative, upregulates autophagy and synergistically sensitizes human GBM8901 glioblastoma cells to temozolomide.

Glioblastoma multiforme (GBM) is the most fatal form of human brain cancer. Although temozolomide (TMZ), an oral alkylating chemotherapeutic agent, improves the survival rate, the prognosis of patients with GBM remains poor. Naturally occurring carbazole alkaloids isolated from curry leaves (Murraya koenigii Spreng.) have been shown to possess a wide range of anticancer properties. However, the effects of carbazole derivatives on glioblastoma cells remain poorly understood. In the present study, anti-glioblastoma profiles of a series of synthetic carbazole derivatives were evaluated in vitro. The most promising derivative in this series was BC3EE2,9B, which showed significant anti-proliferative effects in GBM8401 and GBM8901 cells. BC3EE2,9B also triggered cell-cycle arrest, most prominently at the G1 stage, and suppressed glioblastoma cell invasion and migration. Furthermore, BC3EE2,9B induced autophagy-mediated cell death and synergistically sensitized GBM cells to TMZ cytotoxicity. The possible mechanism underlying BC3EE2,9B-induced autophagy may involve activation of adenosine monophosphate-activated protein kinase and the attenuation of the Akt and mammalian target of the rapamycin downstream signaling pathway. Taken together, the present results provide molecular evidence for the mode of action governing the ability of BC3EE2,9B to sensitize drug-resistant glioblastoma cells to the chemotherapeutic agent TMZ.

Abstract 672: ABTL0812, a new antitumor drug that inhibits the axis Akt/mTOR through a novel mechanism of action

Abstract Background: ABTL0812 is a first-in-class orally administered compound currently in Phase I/Ib First in Human Clinical Trial in patients with advanced solid tumors (NCT02201823). ABTL0812 has cytotoxic effect on a wide range of human tumor cell lines, including those which have become resistant to standard therapy. We hereby dissect the anti-tumor activity of ABTL0812, which relies on a novel mechanism of action that promotes inhibition of the Akt/mTOR axis in cancer cells. Material & methods: ABTL0812 molecular targets were identified by in silico analysis, comparing ABTL0812 chemical structure against a database including more than one million receptor-ligand interaction data. Functional relevance of the targets was confirmed biochemically and pharmacologically. ABTL0812 mechanism of action was established using human lung and pancreatic tumor cells, MEF KO cells, as well as tumor xenografts. Results: In silico screening showed that ABTL0812 binds four targets which regulate tumor progression through Akt/mTOR axis. Two of them are the transcription factors PPARα and PPARγ (Peroxisome-Proliferator Activating Receptors). In lung and pancreatic tumor cells ABTL0812 activated PPARα/γ-dependent gene transcription, while pharmacological inhibition with PPARα/γ antagonists impaired ABTL0812 cytotoxic effect. Interestingly, ABTL0812 induced transcription of the endogenous Akt inhibitor TRIB3 (tribbles homologue 3) through PPARα/γ activation. TRIB3 is a pseudokinase that inhibits Akt by direct binding and preventing its phosphorylation by mTORC2 complex. According to this, ABTL0812-induced TRIB3 overexpression resulted in inhibition of Akt phosphorylation, impaired phosphorylation of the Akt substrates TSC2 and PRAS40 and mTORC1 inhibition (pS6), which in turn promoted autophagy-mediated tumor cell death. MEF TRIB3-/- cells were resistant to ABTL0812-induced cell death, indicating that TRIB3 mediates ABTL0812 citotoxicity. Finally, Akt inhibition was observed in human lung and pancreatic tumor xenograft models treated with ABTL0812 and in human platelets incubated with ABTL0812. This supported the rational for using Akt phosphorylation as a pharmacodynamic biomarker to monitor activity of ABTL0812 in patients included in the Clinical Trial. Conclusion: ABTL0812 promotes autophagy-mediated cell death in different cancer paradigms by inhibiting the Akt/mTOR axis through a novel mechanism of action. ABTL0812 induces PPARα/γ-mediated transcription of the TRIB3 gene. Upregulated TRIB3 protein binds and inhibits Akt, leading to mTORC1 inhibition and reduction of tumor growth. These findings provide evidences that ABTL0812 may be an effective therapeutic strategy for targeting cancer. Citation Format: Tatiana Erazo, Mariana Gomez-Ferreria, Jose Alfon, Mar Lorente, Maria Salazar, Anna Lopez, Marc Cortal, Pau Munoz-Guardiola, Pedro Gascon, Guillermo Velasco, Carles Domenech, Jose M. Lizcano. ABTL0812, a new antitumor drug that inhibits the axis Akt/mTOR through a novel mechanism of action. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 672. doi:10.1158/1538-7445.AM2015-672

The use of cannabinoids as anticancer agents

It is well-established that cannabinoids exert palliative effects on some cancer-associated symptoms. In addition evidences obtained during the last fifteen years support that these compounds can reduce tumor growth in animal models of cancer. Cannabinoids have been shown to activate an ER-stress related pathway that leads to the stimulation of autophagy-mediated cancer cell death. In addition, cannabinoids inhibit tumor angiogenesis and decrease cancer cell migration. The mechanisms of resistance to cannabinoid anticancer action as well as the possible strategies to develop cannabinoid-based combinational therapies to fight cancer have also started to be explored. In this review we will summarize these observations (that have already helped to set the bases for the development of the first clinical studies to investigate the potential clinical benefit of using cannabinoids in anticancer therapies) and will discuss the possible future avenues of research in this area.

Fumonisin B1 induces autophagic cell death via activation of ERN1-MAPK8/9/10 pathway in monkey kidney MARC-145 cells.

Mycotoxins are secondary fungal metabolites that are capable of inducing a variety of toxic effects in animals and humans resulting from the consumption of the contaminated food. Understanding the mechanisms of the toxicities behind these mycotoxins is required to develop mechanism-based approach to counteract their toxic potential. Fumonisin B1 (FB1) is the most prevalent member of fumonisins that are a group of mycotoxins produced primarily by Fusarium verticillioides and Fusarium proliferatum. Kidney is one of the primary target organs for FB1 action. Using monkey kidney MARC-145 cells as an intro model, we found that FB1 induced caspase-independent programmed cell death accompanied with autophagy induction. Inhibition of autophagy by either chemical inhibitors or RNAi approach led to a significant reduction in cell death by FB1 exposure, indicating possible involvement of autophagy-mediated cell death in nephrotoxicity of FB1. Further mechanistic investigation revealed that activation of ERN1-MAPK8/9/10 axis played a critical role in autophagy induction and autophagy-mediated cell death by FB1 exposure. In addition, we demonstrated that disruption of sphingolipid metabolism was an apical event in FB1-induced ERN1-MAPK8/9/10-mediated autophagic cell death in MARC-145 cells. Lastly, we identified curcumin, a naturally occurring plant phenolic compound, as a possible anti-FB1 agent that can be used to protect kidney cells from FB1-induced cell death through inhibition of MAPK8/9/10 activation.