Identification of novel bivalent mimetics of annonaceous acetogenins via a scaffold-hopping strategy

A series of novel bivalent mimetics of annonaceous acetogenins have been designed, synthesized, and evaluated. Among these, compound 7 bearing a homopiperazine ring in the middle region exhibited more potent growth inhibitory activity and higher selectivity against cancer cells over normal cells by comparison with AA005. This work indicates that modification of the middle piperazine ring is a useful optimizing tool for the simplified acetogenin mimetics.     

Identification of an Annonaceous Acetogenin Mimetic,AA005, as an AMPK Activator and Autophagy Inducer in Colon Cancer Cells

Annonaceous acetogenins, a large family of naturally occurring polyketides isolated from various species of the plant genus Annonaceae, have been found to exhibit significant cytotoxicity against a variety of cancer cells. Previous studies showed that these compounds could act on the mitochondria complex-I and block the corresponding electron transport chain and terminate ATP production. However, more details of the mechanisms of action remain ambiguous. In this study we tested the effects of a set of mimetics of annonaceous acetogenin on some cancer cell lines, and report that among them AA005 exhibits the most potent antitumor activity. AA005 depletes ATP, activates AMP-activated protein kinase (AMPK) and inhibits mTOR complex 1 (mTORC1) signal pathway, leading to growth inhibition and autophagy of colon cancer cells. AMPK inhibitors compound C and inosine repress, while AMPK activator AICAR enhances, AA005-caused proliferation suppression and subsequent autophagy of colon cancer cells. AA005 enhances the ATP depletion and AMPK activation caused by 2-deoxyglucose, an inhibitor of mitochondrial respiration and glycolysis. AA005 also inhibits chemotherapeutic agent cisplatin-triggered up-regulation of mTOR and synergizes with this drug in suppression of proliferation and induction of apoptosis of colon cancer cells. These data indicate that AA005 is a new metabolic inhibitor which exhibits therapeutic potentials in colon cancer.     

Evaluation of various parts of the paw paw tree, Asimina triloba (Annonaceae), as commercial sources of the pesticidal annonaceous acetogenins.

Various plant parts of the paw paw tree (Asimina triloba Dunal, Annonaceae) were extracted and partitioned to concentrate the mixture of acetogenins into a standardized pesticidal extract (F005). A bioassay with brine shrimp larvae (Artemia salina Leach) was used to determine the relative potencies of the various extracts. The small twigs (0-0.5 cm diameter) yielded the most potent extract (LC50 = 0.04 ppm); the stem wood (LC50 = 4.9 ppm) and leaves (LC50 = 53.7 ppm) yielded the poorest activities. The unripe fruits, seeds, root wood, root bark, and stem bark were notably potent and, generally, yielded > 2% of their dry weight as F005. The smaller diameter stems were more potent than the larger stems. We conclude that, by pollarding the trees, the entire twigs and small branches of paw paw could be processed to produce a potent acetogenin mixture; this biomass could be made available in quantities needed for commercialization of the pesticidal product and could be renewable through regrowth from the parent trunk and larger branches.     

Inhibition of phorbol ester-stimulated arachidonic acid release by alkylglycerols

Although synthetic analogs of alkylglycerol (AG), such as dodecylglycerol, possess potent biological activities, their mechanism of action has not been determined. We recently detected substantial amounts of AG in unstimulated MDCK cells (Warne, T.R. and Robinson, M. (1991) Anal. Biochem. 198, 302–307) raising the possibility mediator. In this study, we examined the effects of synthetic AG on the release of arachidonic acid and arachidonate metabolites (AA) from Madin Darby canine kidney (MDCK) cells in response to 12-O-tetradecanoylphorbol-13-acetate (TPA) in order to characterize its effects on this signalling pathway. Treatment of MDCK with AG potently inhibited the release of AA during subsequent stimulation with TPA. Dodecylglycerol, the most effective of a series of alkylgycerols tested, was active at concentrations as low as 3 μM. The sn-1 and sn-3 forms of AG were found to be equally potent inhibitors. The effects of AG on AA release were not the result of arachidonic acid redistribution among cellular lipids and were independent of the phospholipid source of the released AA. AG did not inhibit the release of AA from MDCK cells when bradykinin was used as a stimulus, indicating selectivity for the effects produced by phorbol esters. These results show that AG can function as a potent and specific inhibitor of TPA-mediated AA release. The ability of AG to regulate this signalling pathway in intact MDCK cells, together with its natural occurrence, suggests a potential bioregulatory role for the endogenous compound as an inhibitor of protein kinase C.     

Design,synthesis and biological evaluation of 2-indolinone derivatives as PAK1 inhibitors in MDA-MB-231 cells

P21-activated kinase 1 (PAK1) plays a vital role in the proliferation, survival and migration of cancer cells, which has emerged as a promising drug target for cancer therapy. In this study, a series of 2-indolinone derivatives were designed and synthesized through a structure-based strategy. A potent PAK1 inhibitor (ZMF-005) was discovered, which presented an IC₅₀ value of 0.22 μM against PAK1 with potent antiproliferative activity. Furthermore, we predicted the binding mode of ZMF-005 and PAK1 by molecule docking and dynamic (MD) simulation. In addition, ZMF-005 was documented to induce significant apoptosis and suppress migration in MDA-MB-231 cells. Collectively, these findings revealed that ZMF-005 is a novel potent PAK1 inhibitor for breast cancer treatment.     

Synthesis of isoflavene-thiosemicarbazone hybrids and evaluation of their anti-tumor activity

Phenoxodiol is an isoflavene with potent anti-tumor activity. In this study, a series of novel mono- and di-substituted phenoxodiol-thiosemicarbazone hybrids were synthesized via the condensation reaction between phenoxodiol with thiosemicarbazides. The in vitro anti-proliferative activities of the hybrids were evaluated against the neuroblastoma SKN-BE(2)C, the triple negative breast cancer MDA-MB-231, and the glioblastoma U87 cancer cell lines. The mono-substituted hybrids exhibited potent anti-proliferative activity against all three cancer cell lines, while the di-substituted hybrids were less active. Selected mono-substituted hybrids were further investigated for their cytotoxicity against normal MRC-5 human lung fibroblast cells, which identified two hybrids with superior selectivity for cancer cells over normal cells as compared to phenoxodiol. This suggests that mono-substituted phenoxodiol-thiosemicarbazone hybrids have promising potential for further development as anti-cancer agents.     

Essential structural features of acetogenins: role of hydroxy groups adjacent to the bis-THF rings

The presence of two hydroxy groups adjacent to the THF ring(s) is a common structural feature of natural acetogenins. To elucidate the role of each hydroxy group in the inhibitory action of acetogenins, we synthesized three acetogenin analogues which lack either or both of the hydroxy groups, and investigated their inhibitory activities with bovine heart mitochondrial complex I. Our results indicate that the presence of either of the two hydroxy groups sufficiently sustains a potent inhibitory effect.     

Involvement of caspase-3 activation in squamocin-induced apoptosis in leukemia cell line HL-60

Annonaceous acetogenins have potent antitumor effect in vitro and in vivo. Squamocin is one of the annonaceous acetogenins and has been reported to have antiproliferative effect on cancer cells. Our results from this study showed that squamocin inhibited proliferation of HL-60 cells with IC50 value of 0.17 microg/ml and induced apoptosis of HL-60 cells. Investigation of the mechanism of squamocin-induced apoptosis revealed that treatment of HL-60 cells with squamocin resulted in extensive nuclear condensation. DNA fragmentation, cleavage of the death substrate poly (ADP-ribose) polymerase (PARP) and induction of caspase-3 activity. Pretreatment of HL-60 cells with caspase-3 specific inhibitor DEVD-CHO prevented squamocin-induced DNA fragmentation, PARP cleavage and cell death. The expression levels of protein bcl-2, bax have no change in response to squamocin treatment in HL-60 cells, whereas stress-activated protein kinase (SAPK/JNK) was activated after treatment with squamocin in HL-60 cells. These results suggest that apoptosis of HL-60 cells induced by squamocin requires caspase-3 activation and is related to SAPK activation.     

Synthesis of novel isoflavene–propranolol hybrids as anti-tumor agents

Isoflavene–propranolol hybrid molecules were developed as potentially novel anti-tumour agents. Isoflavene itself has potent anti-cancer activity while propranolol can enhance anti-proliferative and anti-angiogenic properties of 5-fluorouracil and paclitaxel. The hybrids were produced via nucleophilic addition of substituted amine groups to a dioxiran intermediate, which was in turn generated from the Williamson-type reaction of isoflavene with (±)-epichlorohydrin. These analogues were tested in anti-cancer cell viability assays against SHEP neuroblastoma and MDA-MB-231 breast adenocarcinoma cell lines, and were found to exhibit potent anti-proliferative activities. These compounds also displayed anti-angiogenic and anti-proliferative effects in HMEC-1 human microvascular endothelial cell lines. Notably, the most potent hybrid molecules synthesized in this work showed enhanced potency against cancer cell lines compared to either isoflavene or propranolol alone, while retaining significant selectivity for cancer cells over MRC-5 normal lung fibroblast cells.     

Antitumor activity of annonaceous acetogenins in HepS and S180 xenografts bearing mice

Annonaceous acetogenins (ACGs), as one of the most powerful groups of mitochondrial complex I inhibitors, exhibit potent cytotoxic activity against a variety of human tumor cell lines. In this study, the antitumor activities of three main types of ACGs were investigated using S180 and HepS xenografts bearing mice simultaneously. The results revealed that select ACGs suppressed tumor growth in a dose-dependent fashion. Tested ACGs showed more selective antitumor activity against HepS. Furthermore, adjacent bis-THF ACGs were more active than mono-THF and nonadjacent bis-THF ACGs against HepS and S180; nonadjacent bis-THF ACGs were more active than mono-THF ACGs against S180, but mono-THF ACGs were more potent than nonadjacent bis-THF ACGs against HepS.     

Synthesis and antiproliferative activity of 2-aryl-4-oxo-thiazolidin-3-yl-amides for prostate cancer

We have previously described serine amide phosphates (SAPs) as a novel class of cytotoxic agents for prostate cancer. Several of them showed potent cytotoxicity against human prostate cancer cell lines, but were not selective in non-tumor cells. To improve the selectivity and further enhance the potency, we designed a new series of 2-aryl-4-oxo-thiazolidin-3-yl amides. The current work describes synthesis, SAR, and biological evaluation of these compounds for their ability to inhibit the growth of prostate cancer cells. The antiproliferative effects of synthesized compounds were examined in five human prostate cancer cell lines (DU-145, PC-3, LNCaP, PPC-1, and TSU), and in RH7777 cells (negative controls). From this study, three potent compounds (8, 20, and 21) have been detected, which are effective in killing prostate cancer cells with improved selectivity compared to SAPs.     

Synthesis and inhibitory activity of ubiquinone-acetogenin hybrid inhibitor with bovine mitochondrial complex I

To elucidate the inhibitory action of acetogenins, the most potent inhibitors of mitochondrial complex I, we synthesized an acetogenin analogue which possesses a ubiquinone ring (i.e., the physiological substrate of complex I) in place of the alpha,beta-unsaturated gamma-lactone ring of natural acetogenins, and named it Q-acetogenin. Our results indicate that the gamma-lactone ring of acetogenins is completely substitutable with the ubiquinone ring. This fact is discussed in light of the inhibitory action of acetogenins.     

Gamma-lactone-Functionalized antitumoral acetogenins are the most potent inhibitors of mitochondrial complex I

To study the relevance of the terminal alpha,beta-unsaturated gamma-methyl-gamma-lactone moiety of the antitumoral acetogenins of Annonaceae for potent mitochondrial complex I inhibition, we have prepared a series of semisynthetic acetogenins with modifications only in this part of the molecule, from the natural rolliniastatin-1 (1) and cherimolin-1 (2). Some of the hydroxylated derivatives (1b, 1d and 1e) in addition to two infrequent natural beta-hydroxy gamma-methyl gamma-lactone acetogenins, laherradurin (3) and itrabin (4), are more potent complex I inhibitors than any other known compounds.     

Analysis of cytotoxic activity at short incubation times reveals profound differences among Annonaceus acetogenins, inhibitors of mitochondrial Complex I

Annonaceous acetogenins are potent cytotoxic agents against tumor cell lines as well as potent inhibitors of mitochondrial Complex I (Degli Esposti and Ghelli Biochim Biophys Acta 1187:116–120, 1994; Degli Esposti et al. Biochem J 301(Pt 1):161–167, 1994; Tormo et al. Arch Biochem Biophys 369:119–126, 1999). Eighteen different ACGs belonging to seven structural sub-families were tested against six tumor and two non tumor cell lines in a MTT cytotoxicity assay to evaluate the correlation between mitochondrial Complex I inhibition and cytotoxic activity potency and selectivity. The results showed a substantial heterogeneity in potency and selectivity among the different compounds tested, although no clear overall structure-activity relationships could be established. To further characterize the biological activity of these compounds, four ACGs were selected based on their inhibition binding sites to Complex I, to evaluate their cytotoxic activity over a 15-minute to 48-hour period using a more sensitive time-course LDH cytotoxicity assay. Our results indicate that, although all of the ACGs were highly cytotoxic in HepG2 cell lines at 24 h, each sub-class behaves rather differently at shorter times. Perhaps other aspects related to how these compounds reach or bind to their target sites, or differences in their ability to cross the cell and/or the mitochondrial membranes, could help explain their different activities. This different behavior between ACGs may provide new clues for a better understanding of their potential antitumor properties.     

High levels of arachidonic acid and peroxisome proliferator-activated receptor-alpha in breast cancer tissues are associated with promoting cancer cell proliferation

Fatty acids are endogenous ligands of peroxisome proliferator-activated receptor-alpha (PPARα), which is linked to the regulation of fatty acid uptake, lipid metabolism and breast cancer cell growth. This study was designed to screen candidate fatty acids from breast cancer tissue and to investigate the effects of these candidate fatty acids on PPARα expression, cell growth and cell cycle progression in breast cancer cell lines. One breast cancer tissue and one reference tissue were each taken from 30 individual breasts to examine for fatty acid composition and PPARα expression. The cancer cell lines MDA-MB-231 (ER–), MCF-7 (ER++++) and BT-474 (ER++) were used to explore the mechanisms regulating cell proliferation. We found that arachidonic acid (AA) and PPARα were highly expressed in the breast cancer tissues. AA stimulated the growth of all three breast cancer cells in a time- and dose-dependent manner. The growth stimulatory effect of AA was associated with PPARα activation, and the most potent effect was found in MCF-7 cells. The stimulation of cell proliferation by AA was accompanied by the increased expression of cyclin E, a reduced population of G1 phase cells, and a faster G1/S phase transition. In contrast, AA had no effects on the levels of CDK2, CDK4, cyclin D1, p27, Bcl-2 and Bax. Our results demonstrate that high levels of AA and PPARα expression in human breast cancer tissues are associated with ER-overexpressed breast cancer cell proliferation, which is involved in activating PPARα, stimulating cyclin E expression, and promoting faster G1/S transition.     

Three new anti-proliferative Annonaceous acetogenins with mono-tetrahydrofuran ring from graviola fruit (Annona muricata)

Bioassay-guided fractionation of the fruit powder of graviola (Annona muricata) yielded three novel compounds: muricins J, K, and L. The compounds are all C35 Annonaceous acetogenins with a mono-tetrahydrofuran ring and four hydroxyls. Their structures were elucidated by spectral methods and chemical modification after isolation via chromatographic techniques and HPLC purification. These three acetogenins demonstrated an antiproliferative against human prostate cancer PC-3 cells.     

Designed antimicrobial and antitumor peptides with high selectivity

We report a new class of cationic amphiphilic peptides with short sequences, G(IIKK)(n)I-NH(2) (n = 1-4), that can kill Gram-positive and Gram-negative bacteria as effectively as several well-known antimicrobial peptides and antibiotics. In addition, some of these peptides possess potent antitumor activities against cancer cell lines. Moreover, their hemolytic activities against human red blood cells (hRBCs) remain remarkably low even at some 10-fold bactericidal minimum inhibitory concentrations (MICs). When bacteria or tumor cells are cocultured with NIH 3T3 fibroblast cells, G(IIKK)(3)I-NH(2) showed fast and strong selectivity against microbial or tumor cells, without any adverse effect on NIH 3T3 cells. The high selectivity and associated features are attributed to two design tactics: the use of Ile residues rather than Leu and the perturbation of the hydrophobic face of the helical structure with the insertion of a positively charged Lys residue. This class of simple peptides hence offers new opportunities in the development of cost-effective and highly selective antimicrobial and antitumor peptide-based treatments.     

Synthesis and cytotoxicity of 4'-C- and 5'-C-substituted toyocamycins

Toyocamycin and some analogues have shown potent antitumor activities; however, none of them could be used clinically primarily owing to their cytotoxicity to normal human cells. In order to overcome the weakness of these nucleoside analogues, substitution of a variety of modified sugars for the ribofuranose was explored in our laboratories with expectation that certain sugar-modified toyocamycin analogues may be selectively cytotoxic to cancer cells. In this article, we report synthesis and cytotoxicity of 4'-C- and 5'-C-substituted toyocamycins, which were prepared via the condensations of 4-C- and 5-C-substituted ribofuranose derivatives 11, 12, 13, 20, 21, and 26 with the silylated form of 4-amino-6-bromo-5-cyanopyrrolo[2,3-]pyrimidine (27) and subsequent debromination and debenzoylation. When compared to the parent toyocamycin, all these analogues showed much lower cytotoxicity to human prostate cancer cells (HTB-81), mouse melanoma cancer cells (B16) as well as normal human fibroblasts. Compound 1e showed a significant cytotoxicity to the prostate cancer cells and a moderate selectivity. The results suggested that sugar modifications, especially those that may affect phosphorylation of nucleosides, could alter cytotoxicity profile significantly.     

Design syntheses and mitochondrial complex I inhibitory activity of novel acetogenin mimics.

Some natural acetogenins are the most potent inhibitors of mitochondrial complex I. These compounds are characterized by two functional units [i.e. hydroxylated tetrahydrofuran (THF) and alpha, beta-unsaturated gamma-lactone ring moieties] separated by a long alkyl spacer. To elucidate which structural factors of acetogenins, including their active conformation, are crucial for the potent inhibitory activity we synthesized a novel bis-acetogenin and its analogues possessing two gamma-lactone rings connected to bis-THF rings by flexible alkyl spacers. The inhibitory potency of the bis-acetogenin with bovine heart mitochondrial complex I was identical to that of bullatacin, one of the most potent natural acetogenins. This result indicated that one molecule of the bis-acetogenin does not work as two reactive inhibitors, suggesting that a gamma-lactone and the THF ring moieties act in a cooperative manner on the enzyme. In support of this, either of the two ring moieties synthesized individually showed no or very weak inhibitory effects. Moreover, combined use of the two ring moieties at various molar ratios exhibited no synergistic enhancement of the inhibitory potency. These observations indicate that both functional units work efficiently only when they are directly linked by a flexible alkyl spacer. Therefore, some specific conformation of the spacer must be important for optimal positioning of the two units in the enzyme. Furthermore, the alpha,beta-unsaturated gamma-lactone, the 4-OH group in the spacer region, the long alkyl tail attached to the THF unit and the stereochemistry surrounding the hydroxylated bis-THF rings were not crucial for the activity, although these are the most common structural features of natural acetogenins. The present study provided useful guiding principles not only for simplification of complicated acetogenin structure, but also for further wide structural modifications of these molecules.     

Ascorbic acid suppresses cell death in rat DS-sarcoma cancer cells induced by 5-aminolevulinic acid-based photodynamic therapy

Especially in the public, vitamin C is considered supportive for the treatment of cancer and supplementation is common. However, the underlying mechanism that most chemotherapeutic agents, ionizing radiation, and photodynamic therapy exert on tumor cell kill is an increased production of reactive oxygen species (ROS) leading to irreversible tissue injury. Therefore, antioxidants like ascorbic acid (AA) may prevent cancer cells of cellular free radical damage and may therefore be contraindicated in patients undergoing tumor treatment. We report on the effects of AA on markers of oxidative stress and apoptosis in rat DS-sarcoma cells on 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT). AA dose-dependently protected cancer cells against lipid and protein oxidation caused by ALA-PDT treatment. By real-time RT-PCR analysis an impressive increase of FasL (124-fold) and TNF-alpha (121-fold) mRNA was detected after PDT treatment. In addition, a decrease in mitochondrial transmembrane potential followed by the mitochondrial release of apoptosis-inducing factor (AIF) was observed. All these early signs of apoptosis were significantly reduced by AA, resulting in a 2.1-fold increased cell survival rate on ALA-PDT treatment. In conclusion, AA functions as a potent antioxidant, protecting mitochondria and other cell structures of oxidative cell injury induced by ALA-PDT and may therefore be contraindicated in patients undergoing tumor treatment.