Inhibition of acyl-CoA binding protein (ACBP) by means of a GABAARγ2-derived peptide.

Acyl-CoA binding protein (ACBP) encoded by diazepam binding inhibitor (DBI) is an extracellular inhibitor of autophagy acting on the gamma-aminobutyric acid A receptor (GABAAR) γ2 subunit (GABAARγ2). Here, we show that lipoanabolic diets cause an upregulation of GABAARγ2 protein in liver hepatocytes but not in other major organs. ACBP/DBI inhibition by systemically injected antibodies has been demonstrated to mediate anorexigenic and organ-protective, autophagy-dependent effects. Here, we set out to develop a new strategy for developing ACBP/DBI antagonists. For this, we built a molecular model of the interaction of ACBP/DBI with peptides derived from GABAARγ2. We then validated the interaction between recombinant and native ACBP/DBI protein and a GABAARγ2-derived eicosapeptide (but not its F77I mutant) by pull down experiments or surface plasmon resonance. The GABAARγ2-derived eicosapeptide inhibited the metabolic activation of hepatocytes by recombinant ACBP/DBI protein in vitro. Moreover, the GABAARγ2-derived eicosapeptide (but not its F77I-mutated control) blocked appetite stimulation by recombinant ACBP/DBI in vivo, induced autophagy in the liver, and protected mice against the hepatotoxin concanavalin A. We conclude that peptidomimetics disrupting the interaction between ACBP/DBI and GABAARγ2 might be used as ACBP/DBI antagonists. This strategy might lead to the future development of clinically relevant small molecules of the ACBP/DBI system.

Guanidine Derivatives Containing the Chalcone Skeleton Are Potent Antiproliferative Compounds against Human Leukemia Cells.

In this study, we investigated the effects of eleven synthetic guanidines containing the 1,3-diphenylpropenone core on the viabilities of six human cancer cells. The most cytotoxic compound against human cancer cells of this series contains a N-tosyl group and a N-methylpiperazine moiety 6f. It was cytotoxic against leukemia cells (U-937, HL-60, MOLT-3, and NALM-6) with significant effects against Bcl-2-overexpressing U-937/Bcl-2 cells as well as the human melanoma SK-MEL-1 cell line. It exhibited low cytotoxicity against quiescent or proliferating human peripheral blood mononuclear cells. The IC50 value for the leukemia U-937 cells was 1.6 ± 0.6 µM, a similar value to that in the antineoplastic agent etoposide. The guanidine containing a N-phenyl substituent 6i was also as cytotoxic as the guanidine containing the N-tosyl substituent and the N-methylpiperazine group 6f against human U-937 leukemia cells and both synthetic guanidines were potent apoptotic inducers. Cell death was mediated by the activation of the initiator caspase-9 and the executioner caspase-3, and associated with the release of cytochrome c. These synthetic guanidines are potent cytotoxic compounds against several human leukemia cells and even the human melanoma cell line SK-MEL-1 and might be useful in the development of new strategies in the fight against cancer.

Structure-activity relationships reveal a 2-furoyloxychalcone as a potent cytotoxic and apoptosis inducer for human U-937 and HL-60 leukaemia cells.

Synthetic flavonoids with new substitution patterns have attracted attention as potential anticancer drugs. Here, twelve chalcones were synthesized and their antiproliferative activities against five human tumour cells were evaluated. This series of chalcone derivatives was characterized by the presence of an additional aromatic or heterocyclic ring linked by an ether, in the case of a benzyl radical, or an ester or amide functional group in the case of a furoyl radical. In addition, the influence on cytotoxicity by the presence of one or three methoxy groups or a 2,4-dimethoxy-3-methyl system on the B ring of the chalcone scaffold was also explored. The results revealed that the most cytotoxic chalcones contain a furoyl substituent linked by an ester or an amide through the 2'-hydroxy or the 2'-amino group of the A ring of the chalcone skeleton, with IC50 values between 0.2 ±â€¯0.1 µM and 1.3 ±â€¯0.1 µM against human leukaemia cells. The synthetic chalcone 2'-furoyloxy-4-methoxychalcone (FMC) was, at least, ten-fold more potent than the antineoplastic agent etoposide against U-937 cells and displayed less cytotoxicity against human peripheral blood mononuclear cells. Treatment of U-937 and HL-60 cells with FMC induced cell cycle arrest at the G2-M phase, an increase in the percentage of sub-G1 and annexin-V positive cells, the release of mitochondrial cytochrome c, activation of caspase and poly(ADP-ribose) polymerase cleavage. In addition, it inhibited tubulin polymerization in vitro in a concentration dependent manner. Cell death triggered by this chalcone was decreased by the pan-caspase inhibitor z-VAD-fmk and was dependent of the generation of reactive oxygen species. We conclude that this furoyloxychalcone may be useful in the development of a potential anti-leukaemia strategy.

Cancer cell-autonomous overactivation of PARP1 compromises immunosurveillance in non-small cell lung cancer.

BACKGROUND: High activity of poly(ADP-ribose) polymerase-1 (PARP1) in non-small cell lung cancer (NSCLC) cells leads to an increase in immunohistochemically detectable PAR, correlating with poor prognosis in patients with NSCLC, as well as reduced tumor infiltration by cytotoxic T lymphocytes (CTLs). Intrigued by this observation, we decided to determine whether PARP1 activity in NSCLC cells may cause an alteration of anticancer immunosurveillance. METHODS: Continuous culture of mouse NSCLC cells in the presence of cisplatin led to the generation of cisplatin-resistant PARhigh clones. As compared with their parental controls, such PARhigh cells formed tumors that were less infiltrated by CTLs when they were injected into immunocompetent mice, suggesting a causative link between high PARP1 activity and compromised immunosurveillance. To confirm this cause-and-effect relationship, we used CRISPR/Cas9 technology to knock out PARP1 in two PARhigh NSCLC mouse cell lines (Lewis lung cancer [LLC] and tissue culture number one [TC1]), showing that the removal of PARP1 indeed restored cisplatin-induced cell death responses. RESULTS: PARP1 knockout (PARP1KO) cells became largely resistant to the PARP inhibitor niraparib, meaning that they exhibited less cell death induction, reduced DNA damage response, attenuated metabolic shifts and no induction of PD-L1 and MHC class-I molecules that may affect their immunogenicity. PARhigh tumors implanted in mice responded to niraparib irrespective of the presence or absence of T lymphocytes, suggesting that cancer cell-autonomous effects of niraparib dominate over its possible immunomodulatory action. While PARhigh NSCLC mouse cell lines proliferated similarly in immunocompetent and T cell-deficient mice, PARP1KO cells were strongly affected by the presence of T cells. PARP1KO LLC tumors grew more quickly in immunodeficient than in immunocompetent mice, and PARP1KO TC1 cells could only form tumors in T cell-deficient mice, not in immunocompetent controls. Importantly, as compared with PARhigh controls, the PARP1KO LLC tumors exhibited signs of T cell activation in the immune infiltrate such as higher inducible costimulator (ICOS) expression and lower PD-1 expression on CTLs. CONCLUSIONS: These results prove at the genetic level that PARP1 activity within malignant cells modulates the tumor microenvironment.

Apoptosis Pathways Triggered by a Potent Antiproliferative Hybrid Chalcone on Human Melanoma Cells.

The World Health Organization reported that approximately 324,000 new cases of melanoma skin cancer were diagnosed worldwide in 2020. The incidence of melanoma has been increasing over the past decades. Targeting apoptotic pathways is a potential therapeutic strategy in the transition to preclinical models and clinical trials. Some naturally occurring products and synthetic derivatives are apoptosis inducers and may represent a realistic option in the fight against the disease. Thus, chalcones have received considerable attention due to their potential cytotoxicity against cancer cells. We have previously reported a chalcone containing an indole and a pyridine heterocyclic rings and an α-bromoacryloylamido radical which displays potent antiproliferative activity against several tumor cell lines. In this study, we report that this chalcone is a potent apoptotic inducer for human melanoma cell lines SK-MEL-1 and MEL-HO. Cell death was associated with mitochondrial cytochrome c release and poly(ADP-ribose) polymerase cleavage and was prevented by a non-specific caspase inhibitor. Using SK-MEL-1 as a model, we found that the mechanism of cell death involves (i) the generation of reactive oxygen species, (ii) activation of the extrinsic and intrinsic apoptotic and mitogen-activated protein kinase pathways, (iii) upregulation of TRAIL, DR4 and DR5, (iv) downregulation of p21Cip1/WAF1 and, inhibition of the NF-κB pathway.

Ethanol Enhances Hyperthermia-Induced Cell Death in Human Leukemia Cells.

Ethanol has been shown to exhibit therapeutic properties as an ablative agent alone and in combination with thermal ablation. Ethanol may also increase sensitivity of cancer cells to certain physical and chemical antitumoral agents. The aim of our study was to assess the potential influence of nontoxic concentrations of ethanol on hyperthermia therapy, an antitumoral modality that is continuously growing and that can be combined with classical chemotherapy and radiotherapy to improve their efficiency. Human leukemia cells were included as a model in the study. The results indicated that ethanol augments the cytotoxicity of hyperthermia against U937 and HL60 cells. The therapeutic benefit of the hyperthermia/ethanol combination was associated with an increase in the percentage of apoptotic cells and activation of caspases-3, -8 and -9. Apoptosis triggered either by hyperthermia or hyperthermia/ethanol was almost completely abolished by a caspase-8 specific inhibitor, indicating that this caspase plays a main role in both conditions. The role of caspase-9 in hyperthermia treated cells acquired significance whether ethanol was present during hyperthermia since the alcohol enhanced Bid cleavage, translocation of Bax from cytosol to mitochondria, release of mitochondrial apoptogenic factors, and decreased of the levels of the anti-apoptotic factor myeloid cell leukemia-1 (Mcl-1). The enhancement effect of ethanol on hyperthermia-activated cell death was associated with a reduction in the expression of HSP70, a protein known to interfere in the activation of apoptosis at different stages. Collectively, our findings suggest that ethanol could be useful as an adjuvant in hyperthermia therapy for cancer.

Chlorinated Guaiane-Type Sesquiterpene Lactones as Cytotoxic Agents against Human Tumor Cells.

Guaiane-type sesquiterpene lactones are naturally occurring compounds which have attracted attention due to their array of biological activities. In this study, chlorinated guaianolides 1-8, isolated from plants of the genus Centaurea, were evaluated against the human leukemia cell lines HL-60, U-937, a specific U-937 cell line that overexpresses the anti-apoptotic Bcl-2 protein and the human melanoma cell line SK-MEL-1. This established the relevant structure-growth inhibition relationships. Chlorohyssopifolins A (1), C (3) and D (4) and linichlorin A (6) were the most potent compounds in terms of inducing growth inhibition in the four cell lines. IC50 values were below 10 µM in all cases. Chlorohyssopifolins A (1) and D (4) and linichlorin A (6) were potent apoptotic inducers in human U-937 leukemia cells, as determined by fluorescent microscopy and flow cytometry, and their mechanism of action was associated with cytochrome c release, caspase activation and poly(ADP-ribose)polymerase cleavage. Overall this study shows that guaianolides induce cytotoxicity against human tumor cells and provides important insights into the cell death pathways that are involved.

Cytotoxicity of the Sesquiterpene Lactone Spiciformin and Its Acetyl Derivative against the Human Leukemia Cell Lines U-937 and HL-60.

Spiciformin (1) is a sesquiterpene lactone with a germacrane skeleton that is found in two Tanacetum species endemic to the Canary Islands. In this study, the cytotoxicities of 1 and its acetyl derivative (2) were evaluated against human tumor cells. These sesquiterpene lactones were cytotoxic against human acute myeloid leukemia (U-937 and HL-60) cells, even in cells over-expressing the pro-survival protein Bcl-2, but melanoma (SK-MEL-1) and human mononuclear cells isolated from blood of healthy donors were more resistant. Both compounds are apoptotic inducers in human leukemia U-937 cells. Cell death was mediated by the processing and activation of initiator and effector caspases and the cleavage of poly(ADP-ribose) polymerase, and it was blocked by a broad-spectrum caspase inhibitor and (in the case of sesquiterpene lactone 2) by the selective caspase-3/7, -8, and -9 inhibitors. In addition, certainly in the case of compound 2, this was found to be associated with a decrease in mitochondrial membrane potential, downregulation of the anti-apoptotic protein Bcl-2, activation of the mitogen-activated protein kinases signaling pathway, and generation of reactive oxygen species. It will, therefore, be relevant to continue characterization of this class of compounds.

The synthetic flavanone 6-methoxy-2-(naphthalen-1-yl)chroman-4-one induces apoptosis and activation of the MAPK pathway in human U-937 leukaemia cells.

Synthetic flavonoids containing a naphthalene ring have attracted attention as potential cytotoxic compounds. Here, we synthesized ten chalcones and their corresponding flavanones and evaluated their antiproliferative activity against the human tumour cell line U-937. This series of chalcone derivatives was characterized by the presence of a naphthalene ring which was kept unaltered- and attached to the ß carbon of the 1-phenyl-2-propen-1-one framework. The structure-activity relationship of these chalcone derivatives and their corresponding cyclic compounds was investigated by the introduction of different substituents (methyl, methoxy, benzyloxy, chlorine) or by varying the position of the methoxy or benzyloxy groups on the A ring. The results revealed that both the chalcone containing the methoxy group at 5' position of the A ring as well as its corresponding flavanone [6-methoxy-2-(naphthalen-1-yl)chroman-4-one] were the most cytotoxic compounds, with IC50 values of 2.8 ± 0.2 and 1.3 ± 0.2 µM, respectively, against U-937 cells. This synthetic flavanone was as cytotoxic as the antitumor etoposide in U-937 cells and displayed strong cytotoxicity against additional human leukaemia cell lines, including HL-60, MOLT-3 and NALM-6. Human peripheral blood mononuclear cells were more resistant than leukaemia cells to the cytotoxic effects of the flavanone. Treatment of U-937 cells with this compound induced G2-M cell cycle arrest, an increase in sub-G1 ratio and annexin-V positive cells, mitochondrial cytochrome c release, caspase activation and poly(ADP-ribose)polymerase processing. Apoptosis induction triggered by this flavonoid was blocked by overexpression of the anti-apoptotic protein Bcl-2. This flavanone induces phosphorylation of p38 mitogen-activated protein kinases, extracellular-signal regulated kinases and c-jun N-terminal kinases/stress-activated protein kinases (JNK/SAPK) following different kinetics. Moreover, cell death was attenuated by the inhibition of mitogen-activated extracellular kinases and JNK/SAPK and was independent of reactive oxygen species generation.

6'-Benzyloxy-4-bromo-2'-hydroxychalcone is cytotoxic against human leukaemia cells and induces caspase-8- and reactive oxygen species-dependent apoptosis.

In this study, we investigated the effects of synthetic 6'-benzyloxy-4-bromo-2'-hydroxychalcone on viabilities of seven human leukaemia cells. It was cytotoxic against U-937, HL-60, K-562, NALM-6, MOLT-3 cells, and also against Bcl-2-overexpressing U-937/Bcl-2 cells and P-glycoprotein-overexpressing K-562/ADR, but had no significant cytotoxic effects against quiescent or proliferating human peripheral blood mononuclear cells. This chalcone is a potent apoptotic inducer in human leukaemia U-937 cells. Cell death was (i) mediated by the activation and the cleavage of initiator and executioner caspases and poly(ADP-ribose) polymerase; (ii) prevented by the pan-caspase inhibitor z-VAD-fmk, and by the selective caspase-3/7, -6 and -8 inhibitors, and by a cathepsins B/L inhibitor; (iii) associated with the release of mitochondrial proteins, including cytochrome c and Smac/DIABLO; (iv) accompanied by dissipation of the mitochondrial membrane potential, (v) partially blocked by the inhibition of p38MAPK and (vi) mostly abrogated by catalase. In conclusion, the synthetic chalcone is cytotoxic against several types of human leukaemia cell with apoptosis being induced by activation of the extrinsic pathway and the generation of reactive oxygen species.

Gardenin B-induced cell death in human leukemia cells involves multiple caspases but is independent of the generation of reactive oxygen species.

Flavonoids have attracted great interest due to their possible anticancer activities. Here we investigated the antiproliferative activity of the flavonoids isolated from Baccharis scandens against human leukemia cell lines and found that the methoxyflavonoid gardenin B was the most cytotoxic compound against HL-60 and U-937 cells, showing IC50 values between 1.6 and 3.0 µM, but had no significant cytotoxic effects against quiescent or proliferating human peripheral blood mononuclear cells. These effects on viability were accompanied by the concentration- and time-dependent appearance of apoptosis as evidenced by DNA fragmentation, formation of apoptotic bodies and a sub-G1 ratio increase. Comparative studies with the best-studied bioflavonoid quercetin indicate that gardenin B is a more cytotoxic and more apoptotic inducer than quercetin. Cell death induced by gardenin B was associated with: (i) a significant induction of caspase-2, -3, -8 and -9 activities; (ii) cleavage of the initiator caspases (caspase-2, -8 and -9), of the executioner caspase-3, and of poly(ADP-ribose) polymerase; and (iii) a concentration-dependent reactive oxygen species generation. In conclusion, apoptosis induced by gardenin B is associated with activation of both the extrinsic and the intrinsic apoptotic pathways of cell death and occurs through a mechanism that is independent of the generation of reactive oxygen species.