Discovery and SAR of indole-2-carboxylic acid benzylidene-hydrazides as a new series of potent apoptosis inducers using a cell-based HTS assay

A series of indole-2-carboxylic acid benzylidene-hydrazides has been identified as a new class of potent apoptosis inducers through a novel cell-based caspase HTS assay. The screening hit, 5-chloro-3-methyl-indole-2-carboxylic acid (4-nitrobenzylidene)-hydrazide (3a), was found to arrest T47D cells in G(2)/M and to induce apoptosis as measured by the flow cytometric analysis assay. A SAR study was carried out by modification of the substitutions on the indole and benzene rings. Substitution at the 3-position of the indole ring was found to be important for apoptotic activity. A 20-fold increase of apoptotic activity was achieved from screening hit 3a to 5-methyl-3-phenyl-indole-2-carboxylic acid (4-methylbenzylidene)-hydrazide (9a) and 5-chloro-3-phenyl-indole-2-carboxylic acid (4-nitrobenzylidene)-hydrazide (9b), with EC(50) value of 0.1microM in the caspase activation assay in T47D breast cancer cells. Compound 9b also was found to be highly active in a standard growth inhibition assay with a GI(50) value of 0.9microM in T47D cells. Compound 3a and its analogs were found to inhibit tubulin polymerization, which is the most probable primary mechanism of action of these compounds.     

Synthesis and Evaluation of Gambogic Acid Derivatives as Antitumor Agents. Part III

Gambogic acid (GA) has been reported as a potent apoptosis inducer. Previously, we have reported chemical modification at C(34) and C(39) of GA, leading to some agents with improved activity. To investigate the further structure? activity relationship (SAR) and preliminary mechanism of GA activity, a series of derivatives with modified prenyl side chains of GA were synthesized and evaluated. Most of the derivatives showed potent inhibitory activities against the proliferation of HepG2 and A549 cell lines. Compound 4 was selected for further mechanistic studies due to its outstanding activity. It was established that 4 induces the apoptosis of HepG2 cells by using Annexin‐V/PI double staining and Western blot assay, thus, compound 4 can serve as a promising lead compound for the development of novel apoptosis in anticancer treatment.     

Discovery of 1-benzoyl-3-cyanopyrrolo[1,2-a]quinolines as a new series of apoptosis inducers using a cell- and caspase-based high-throughput screening assay. Part 1: Structure-activity relationships of the 1- and 3-positions

1-Benzoyl-3-cyanopyrrolo[1,2-a]quinoline (2a) was identified as a novel apoptosis inducer through our caspase- and cell-based high-throughput screening assay. Compound 2a had good activity against several breast cancer cell lines but was much less active against several other cancer cell lines. SAR studies of 2a found that substitution at the 4-position of the 1-benzoyl group was important for activity. Replacing the 3-cyano group by an ester or ketone group led to inactive compounds. Interestingly, 4-substituted analogs such as 1-(4-(1H-imidazol-1-yl)benzoyl)-3-cyanopyrrolo[1,2-a]quinoline (2k) were found to be broadly and highly active in the caspase activation assay as well as in the cell growth inhibition assay with low nM EC(50) and GI(50) values in human breast cancer cells T47D, human colon cancer cells HCT116, and hepatocellular carcinoma cancer cells SNU398. Compound 2a was found not to inhibit tubulin polymerization up to 50 microM, while 2k was found to inhibit tubulin polymerization with an IC(50) value of 5 microM, indicating that certain substituents at the 4-position of the 1-benzoyl group can change the mechanism of action.     

Gambogic acid induced oxidative stress dependent caspase activation regulates both apoptosis and autophagy by targeting various key molecules (NF-κB,Beclin-1, p62 and NBR1) in human bladder cancer cells

BackgroundGambogic acid is a potent anticancer agent and has been found effective against various types of cancer cells. The present study was addressed to explore the cytotoxic potential of Gambogic acid and the modulation of autophagy and apoptosis in bladder cancer cells T24 and UMUC3.MethodsBladder cancer cell lines T24 and UMUC3 were treated with Gambogic acid, apoptosis was checked by flow-cytometry and expression of various autophagy and apoptosis related proteins was monitored by Western blotting. Confocal microscope was used for colocalization of p62 and Beclin-1.ResultsGambogic acid induces reactive oxygen species, and elicits a strong autophagic response by activating JNK at earlier time points, which is inhibited at later time points with the activation of caspases. Reactive oxygen species mediated caspase activation causes degradation of autophagic proteins, cleavage of molecular chaperones (Hsp90 and GRP-78) and adaptor proteins (p62 and NBR1). Gambogic acid treatment results in mitochondrial hyperpolarization and cytochrome c release and activates caspases involved in both extrinsic and intrinsic apoptotic pathways. Gambogic acid abrogates NF-κB activation by ROS mediated inhibition of IκB-α phosphorylation. Functionally Gambogic acid induced autophagy acts as a strong cell survival response and delays caspase activation.ConclusionOur study provides the new insights about the mechanism of Gambogic acid induced modulation of autophagy and apoptosis in bladder cancer cells. All the molecular events responsible for Gambogic acid induced autophagy and apoptosis are mediated by reactive oxygen species.General significanceSince Gambogic acid targets various cell survival molecules therefore, it may be considered as a potential anticancer agent against bladder cancer.     

Discovery and structure–activity relationships of (2-(arylthio)benzylideneamino)guanidines as a novel series of potent apoptosis inducers

1-(2-(2,5-Dimethoxyphenylthio)benzylidene)semicarbazide (2a) was discovered as a potent apoptosis inducer through our cell based HTS assay. SAR study led to the discovery of a more aqueous soluble analog (2-(2,5-dimethoxyphenylthio)-6-methoxybenzylideneamino)guanidine (5e) with EC₅₀ value of 60 nM in the caspase activation assay and GI₅₀ value of 62 nM in the growth inhibition assay in T47D cells. Compound 5e was found to be an inhibitor of tubulin polymerization and efficacious in a MX-1 breast tumor model.     

Discovery of N-aryl-9-oxo-9H-fluorene-1-carboxamides as a new series of apoptosis inducers using a cell- and caspase-based high-throughput screening assay. 1. Structure–activity relationships of the carboxamide group

N-(2-Methylphenyl)-9-oxo-9H-fluorene-1-carboxamide (2a) was identified as a novel apoptosis inducer through our caspase- and cell-based high-throughput screening assay. Compound 2a was found to be active with sub-micromolar potencies for both caspase induction and growth inhibition in T47D human breast cancer, HCT116 human colon cancer, and SNU398 hepatocellular carcinoma cancer cells. It arrested HCT116 cells in G₂/M followed by apoptosis as assayed by the flow cytometry. Structure–activity relationship (SAR) studies of the carboxamide group identified the lead compound N-(2-(1H-pyrazol-1-yl)phenyl)-9-oxo-9H-fluorene-1-carboxamide (6s). Compound 6s, with increased aqueous solubility, was found to retain the broad activity in the caspase activation assay and in the cell growth inhibition assay with sub-micromolar EC₅₀ and GI₅₀ values in T47D, HCT116, and SNU398 cells, respectively.     

Discovery of substituted 4-anilino-2-(2-pyridyl)pyrimidines as a new series of apoptosis inducers using a cell- and caspase-based high throughput screening assay. Part 1: structure-activity relationships of the 4-anilino group

A series of 4-anilino-2-(2-pyridyl)pyrimidines has been discovered as a new class of potent inducers of apoptosis using a cell-based HTS assay. Compound 5a was found to arrest T47D cells in G2/M and induced apoptosis. SAR studies showed that a small and electron-donating group at the meta-position of the anilino ring is important for activity. A 20-fold increase in potency, from hit compound 4-(3-methoxyanilino)-2-(2-pyridinyl)-6-(trifluoromethyl)pyrimidine (5a) to lead compound 4-(2,5-dimethoxyanilino)-2-(2-pyridinyl)-6-(trifluoromethyl)pyrimidine (5l), was obtained through the SAR studies. Compound 5l is highly active with an EC50 value of 18 nM in the caspase activation assay in T47D breast cells. Interestingly, 5a and other meta-mono-substituted compounds were active against T47D cells but were not active against H1299 and HT29 cells, while 5l and other 2,5-disubstituted compounds were active against all the three cells. In a tubulin polymerization assay, compound 5l inhibited tubulin polymerization with an IC50 value of < 0.5 microM, while 5a was not active up to 50 microM.     

Comparative cellular and molecular analysis of cytotoxicity and apoptosis induction by doxorubicin and <Emphasis Type="Italic">Baneh</Emphasis> in human breast cancer T47D cells

It is now widely accepted that dietary phytochemicals inhibit cancer progression and enhance the effects of conventional chemotherapy. In this report, we comparatively studied the cellular and molecular aspects of apoptosis induction by the methanolic extract of Baneh fruit skin in comparison to Doxorubicin (Dox), a well-known anticancer drug, in human breast cancer T47D cells. The MTT assay was used to determine the antiproliferative effects. The flow cytometric and microscopic analyses were done to evaluate the apoptosis induction. Furthermore, western blot analyses have been done to study the role of key molecular players of apoptosis including caspase 3 and PARP. The Baneh extract showed strong antiproliferative activity against T47D cells in a dose- and time-dependent manner that was comparable to and even stronger than Dox in certain concentrations. Analysis of Baneh-treated cells by flow cytometry and fluorescence microscopy indicated strong apoptosis induction and nuclear morphological alterations similar to or greater than Dox. Finally, molecular analysis of apoptosis by western blotting proved activation of caspase 3 followed by poly ADP ribose polymerase (PARP) cleavage more efficiently in Baneh than in Dox treated cancer cells. These findings indicate that Baneh extract contains phytochemicals which act as inhibitor of cell proliferation and inducer of apoptosis in human breast cancer T47D cells that makes it a potentially good candidate for new anticancer drug development.     

Discovery of 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-phenyl-(E)-2,3,6,7-tetrahydro-1,4-thiazepines as a new series of apoptosis inducers using a cell- and caspase-based HTS assay

We report the discovery of 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-(4-methylphenyl)-(E)-2,3,6,7-tetrahydro-1,4-thiazepine (2a) as an inducer of apoptosis using our proprietary cell- and caspase-based HTS assay. Through structure activity relationship (SAR) studies, 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-(2-methoxy-4-(methylthio)phenyl)-(E)-2,3,6,7-tetrahydro-1,4-thiazepine (5d) was identified as a potent apoptosis inducer with an EC(50) value of 0.08 microM in T47D cells, which was >15-fold more potent than screening hit 2a. Compound 5d also was found to be highly active in a growth inhibition assay with a GI(50) value of 0.05 microM in T47D cells and to function as an inhibitor of tubulin polymerization.     

Interaction of vitamin D analogs with signaling pathways leading to active cell death in breast cancer cells

Induction of apoptosis is a feature of the anti-tumor effects of certain vitamin D analogs. The aim of this study was to identify if common effectors are involved in cell death mediated by serum starvation, vitamin D analogs and tumor necrosis factor (TNF) alpha in 3 human breast cancer cell lines: MCF-7, T47-D and Hs578T. Incubation of cells in serum-free medium induced apoptosis as assessed by loss of cell viability and increased DNA fragmentation. Addition of IGF-I (30 ng/ml) protected against loss of cell viability in MCF-7 cells and co-treatment with two synthetic analogs (CB1093 and EB1089, 50 nM for 4 days) prevented these anti-apoptotic effects of IGF-I. Pretreatment of MCF-7 and Hs578T cells with the vitamin D analogs substantially potentiated the cytotoxic effects of TNFalpha. This cytokine was not cytotoxic for T47-D cells but co-incubation with CB1093 led to loss of cell viability. Potentiation by CB1093 of TNFalpha-induced apoptosis in MCF-7 cells was accompanied by increased activation of cytosolic phospholipase A2 and arachidonic acid release, which was partially inhibited by AACOCF3, a specific cPLA2 inhibitor. The broad-spectrum caspase inhibitor z-VAD-fmk prevented TNFalpha but not CB1093 mediated cell death and activation of cPLA2. Serum starvation induced apoptosis was accompanied by cPLA2 activation, which was inhibited by IGF-I and by z-VAD-fmk. However, the ability of these agents to suppress cPLA2 activation was abrogated by co-treatment with CB1093, suggesting a role for arachidonic acid release in the caspase-independent mechanism by which vitamin D analogs prevent the protective effects of IGF-I on breast cancer cell survival.     

Studies on chemical structure modification and structureactivity relationship of derivatives of gambogic Acid at c(39)

The natural product gambogic acid exhibits high potency in inhibiting cancer cell lines. Rational medicinal modifications on gambogic acid will improve its physicochemical properties and drug-like characters. To investigate the structure?activity relationship of gambogic acid and also to find rational modification position on its chemical skeleton, we designed, synthesized, and characterized 16 derivatives of gambogic acid that were modified at C(39). The structure?activity relationships (SARs) were discussed. The anti-proliferation data were accquired through MTT (=3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assays of A549, BGC823, U251, HepG2, and MDA-MB-231 cancer cell lines. Most of the synthesized compounds showed strong inhibitory effects. The SAR study revealed that derivatives with aliphatic amino moieties at C(39) were more potent than those with other substituents. The C(39) position can undergo different kinds of chemical modifications without leading to loss of activity. Compounds 4 and 6 can serve as potential lead compounds for further development of new anticancer drugs.     

Structure elucidation of metabolites of gambogic acid in vivo in rat bile by high-performance liquid chromatography-mass spectrometry and high-performance liquid chromatography-nuclear magnetic resonance

Gambogic acid (GBA), the main component of Gamboge, possesses significant anti-tumour activity. Due to its structural complexity, little is known about GBA metabolism. Here, we investigate the metabolism of GBA in vivo in rat bile. Identification of the metabolites formed was elucidated using high-performance liquid chromatography (HPLC) with UV-vis detection, HPLC/ion trap electrospray ionization-mass spectrometry, as well as HPLC/nuclear magnetic resonance. Four main metabolites were determined. Two phase I metabolites, 10-hydroxygambogic acid and 9,10-epoxygambogic acid, were oxides on the 9,10-olefinic bond of GBA. The others phase II metabolites, were 9,10-epoxygambogic acid-30-O-glucuronide and 10-hydroxylgambogic acid-30-O-glucuronide.     

Proliferation and apoptosis in a neuroblastoma cell line exposed to 900 MHz modulated radiofrequency field

The aim of this study was to examine whether a modulated radiofrequency of the type used in cellular phone communications at a specific absorption rate (SAR) higher than International Commission on Non-ionizing Radiation Protection (ICNIRP) reference level for occupational exposure, could elicit alterations on proliferation, differentiation, and apoptosis processes in a neuroblastoma cell line. The cell line was exposed for 24, 48, and 72 h to 900 MHz radiofrequency and proliferation and differentiation were tested by WST-I assay and by a molecular analysis of specific markers, two oncogenes and a cytoskeleton protein, in exponential growth phase and in synchronized cell cultures. Apoptosis was evaluated by caspase activation analysis and by molecular detection of Poly (ADP-ribose) polimerase (PARP) cleavage. Combined exposures to radiofrequency and to the differentiative agent retinoic acid or to the apoptotic inducer camptothecin were carried out to test possible interference between electromagnetic field and chemical agents. Overall our data suggest that 900 MHz radiofrequency exposure up to 72 h does not induce significant alterations in the three principal cell activities in a neuroblastoma cell line.     

Discovery of 1-benzoyl-3-cyanopyrrolo[1,2-a]quinolines as a new series of apoptosis inducers using a cell- and caspase-based high-throughput screening assay. 2: Structure–activity relationships of the 4-, 5-, 6-, 7- and 8-positions

As a continuation of our efforts to discover and develop the apoptosis inducing 1-benzoyl-3-cyanopyrrolo[1,2-a]quinolines as potential anticancer agents, we explored substitutions at the 4-, 5-, 6-, 7- and 8-positions of pyrrolo[1,2-a]quinoline. SAR studies showed that substitution at the 6-position by a small group such as Cl resulted in potent compounds. Substitutions at the 5- and 8-positions were tolerated while substitutions at the 4- and 7-position led to inactive compounds. Several compounds, including 2c, 3a, 3b and 3f, were found to be highly active against human breast cancer cells T47D with EC₅₀ values of 0.053–0.080 μM, but much less active against human colon cancer cells HCT116 and hepatocellular carcinoma cancer cells SNU398 in the caspase activation assay. Compound 3f also was found to be highly active with a GI₅₀ value of 0.018 μM against T47D cells in a growth inhibition assay.     

Studies on Chemical Structure Modification and Structure?Activity Relationship of Derivatives of Gambogic Acid at C(39)

The natural product gambogic acid exhibits high potency in inhibiting cancer cell lines. Rational medicinal modifications on gambogic acid will improve its physicochemical properties and drug‐like characters. To investigate the structure? activity relationship of gambogic acid and also to find rational modification position on its chemical skeleton, we designed, synthesized, and characterized 16 derivatives of gambogic acid that were modified at C(39). The structure? activity relationships (SARs) were discussed. The anti‐proliferation data were accquired through MTT (=3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide) assays of A549, BGC823, U251, HepG2, and MDA‐MB‐231 cancer cell lines. Most of the synthesized compounds showed strong inhibitory effects. The SAR study revealed that derivatives with aliphatic amino moieties at C(39) were more potent than those with other substituents. The C(39) position can undergo different kinds of chemical modifications without leading to loss of activity. Compounds 4 and 6 can serve as potential lead compounds for further development of new anticancer drugs.     

Release of mitochondrial cytochrome C in both apoptosis and necrosis induced by beta-lapachone in human carcinoma cells.

BACKGROUND: There are two fundamental forms of cell death: apoptosis and necrosis. Molecular studies of cell death thus far favor a model in which apoptosis and necrosis share very few molecular regulators. It appears that apoptotic processes triggered by a variety of stimuli converge on the activation of a member of the caspase family, such as caspase 3, which leads to the execution of apoptosis. It has been suggested that blocking of caspase activation in an apoptotic process may divert cell death to a necrotic demise, suggesting that apoptosis and necrosis may share some upstream events. Activation of caspase is preceded by the release of mitochondrial cytochrome C. MATERIALS AND METHODS: We first studied cell death induced by beta-lapachone by MTT and colony-formation assay. To determine whether the cell death induced by beta-lapachone occurs through necrosis or apoptosis, we used the PI staining procedure to determine the sub-G1 fraction and the Annexin-V staining for externalization of phophatidylserine. We next compared the release of mitochondrial cytochrome C in apoptosis and necrosis. Mitochondrial cytochrome C was determined by Western blot analysis. To investigate changes in mitochondria that resulted in cytochrome C release, the mitochondrial membrane potential (delta psi) was analyzed by the accumulation of rhodamine 123, a membrane-permeant cationic fluorescent dye. The activation of caspase in apoptosis and necrosis were measured by using a profluorescent substrate for caspase-like proteases, PhiPhiLuxG6D2. RESULTS: beta-lapachone induced cell death in a spectrum of human carcinoma cells, including nonproliferating cells. It induced apoptosis in human ovary, colon, and lung cancer cells, and necrotic cell death in four human breast cancer cell lines. Mitochondrial cytochrome C release was found in both apoptosis and necrosis. This cytochrome C release occurred shortly after beta-lapachone treatment when cells were fully viable by trypan blue exclusion and MTT assay, suggesting that cytochrome C release is an early event in beta-lapachone induced apoptosis as well as necrosis. The mitochondrial cytochrome C release induced by beta-lapachone is associated with a decrease in mitochondrial transmembrane potential (delta psi). There was activation of caspase 3 in apoptotic cell death, but not in necrotic cell death. This lack of activation of CPP 32 in human breast cancer cells is consistent with the necrotic cell death induced by beta-lapachone as determined by absence of sub-G1 fraction, externalization of phosphatidylserine. CONCLUSIONS: beta-lapachone induces either apoptotic or necrotic cell death in a variety of human carcinoma cells including ovary, colon, lung, prostate, and breast, suggesting a wide spectrum of anti-cancer activity in vitro. Both apoptotic and necrotic cell death induced by beta-lapachone are preceded by a rapid release of cytochrome C, followed by the activation of caspase 3 in apoptotic cell death but not in necrotic cell death. Our results suggest that beta-lapachone is a potential anti-cancer drug acting on the mitochondrial cytochrome C-caspase pathway, and that cytochrome C is involved in the early phase of necrosis.     

Dipeptidyl aspartyl fluoromethylketones as potent caspase inhibitors: peptidomimetic replacement of the P2 alpha-amino acid by a alpha-hydroxy acid

As a continuation of our SAR studies of dipeptidyl aspartyl-fmk as caspase inhibitors, we explored the replacement of the P2 alpha-amino acid by a peptidomimetic alpha-hydroxy acid. These alpha-carbamoyl-alkylcarbonyl-aspartyl fluoromethylketones were found to be potent caspase inhibitors, and the SAR of these compounds is similar to the corresponding dipeptidyl aspartyl-fmk. MX1153, (S)-3-methyl-2-(phenylcarbamoyl)butanoyl-Asp-fmk, is identified as a potent broad-spectrum caspase inhibitor, and is selective for caspases versus other proteases. MX1153 also has good activity in the cell apoptosis protection assays and is active in the mouse liver apoptosis model.     

The role of the Ah receptor and p38 in benzo[a]pyrene-7,8-dihydrodiol and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide-induced apoptosis

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants in the environment. Benzo[a]pyrene (B[a]P), a prototypical member of this class of chemicals, affects cellular signal transduction pathways and induces apoptosis. In this study, the proximate carcinogen of B[a]P metabolism, trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-dihydrodiol) and the ultimate carcinogen, B[a]P-r-7,t-8-dihydrodiol-t-9,10-epoxide(+/-) (BPDE-2) were found to induce apoptosis in human HepG2 cells. Apoptosis initiated by B[a]P-7,8-dihydrodiol was linked to activation of the Ah receptor and induction of CYP1A1, an event that can lead to the formation of BPDE-2. With both B[a]P-7,8-dihydrodiol and BPDE-2 treatment, changes in anti- and pro-apoptotic events in the Bcl-2 family of proteins correlated with the release of mitochondrial cytochrome c and caspase activation. The onset of apoptosis as monitored by caspase activation was linked to mitogen-activated protein (MAP) kinases. Utilizing mouse hepa1c1c7 cells and the Arnt-deficient BPRc1 cells, activation of MAP kinase p38 by B[a]P-7,8-dihydrodiol was shown to be Ah receptor-dependent, indicating that metabolic activation by CYP1A1 was required. This was in contrast to p38 activation by BPDE-2, an event that was independent of Ah receptor function. Confirmation that MAP kinases play a critical role in BPDE-2-induced apoptosis was shown by inhibiting caspase activation of poly(ADP-ribose)polymerase 1 (PARP-1) by chemical inhibitors of p38 and ERK1/2. Furthermore, mouse embryo p38-/- fibroblasts were shown to be resistant to the actions of BPDE-2-induced apoptosis as determined by annexin V analysis, cytochrome c release, and cleavage of PARP-1. These results confirm that the Ah receptor plays a critical role in B[a]P-7,8-dihydrodiol-induced apoptosis while p38 MAP kinase links the actions of an electrophilic metabolite like BPDE-2 to the regulation of programmed cell death.     

Dipeptidyl aspartyl fluoromethylketones as potent caspase inhibitors: peptidomimetic replacement of the P(2) amino acid by 2-aminoaryl acids and other non-natural amino acids

As a continuation of our SAR studies of dipeptidyl aspartyl-fmk as caspase inhibitors, we explored the replacement of the P(2) amino acid by a 2-aminoaryl acid or other non-natural amino acids. Several of these compounds, such as 6l and 6p, were found to have good activities with inhibition potencies of around 100 nM in a caspase-3 enzyme assay. EP1113, Z-Val-(2-aminobenzoyl)-Asp-fmk (9b), is identified as a potent broad-spectrum caspase inhibitor with IC(50) values of 6-60 nM in different caspases. EP1113 also has good activity in a cell apoptosis protection assay.