Apoptosis induction by 13-acetoxyrolandrolide through the mitochondrial intrinsic pathway.

The aim of this study was to evaluate the mechanisms of cytotoxicity of the sesquiterpene lactone 13-acetoxyrolandrolide, a nuclear factor kappa B (NF-κB) inhibitor that was previously isolated from Rolandra fruticosa. The effects associated with the inhibition of the NF-κB pathway included dose-dependent inhibition of the NF-κB subunit p65 (RelA) and inhibition of upstream mediators IKKß and oncogenic Kirsten rat sarcoma (K-Ras). The inhibitory concentration of 13-acetoxyrolandrolide on K-Ras was 7.7 µM. The downstream effects of the inhibition of NF-κB activation were also investigated in vitro. After 24 h of treatment with 13-acetoxyrolandrolide, the mitochondrial transmembrane potential was depolarized in human colon cancer (HT-29) cells. The mitochondrial oxidative phosphorylation was also negatively affected, and reduced levels of nicotinamine adenine dinucleotide phosphate (NAD(P)H) were detected after 2 h of 13-acetoxyrolandrolide exposure. Furthermore, the expression of the pro-apoptotic protein caspase-3 increased in a concentration-dependent manner. Cell flow cytometry showed that 13-acetoxyrolandrolide induced cell cycle arrest at G1 , indicating that the treated cells had undergone caspase-3-mediated apoptosis, indicating negative effects on cancer cell proliferation. These results suggest that 13-acetoxyrolandrolide inhibits NF-κB and K-Ras and promotes cell death mediated through the mitochondrial apoptotic pathway.

Bioactive flavaglines and other constituents isolated from Aglaia perviridis.

Eight new compounds, including two cyclopenta[b]benzopyran derivatives (1, 2), two cyclopenta[b]benzofuran derivatives (3, 4), three cycloartane triterpenoids (5-7), and an apocarotenoid (8), together with 16 known compounds, were isolated from the chloroform-soluble partitions of separate methanol extracts of a combination of the fruits, leaves, and twigs and of the roots of Aglaia perviridis collected in Vietnam. Isolation work was monitored using human colon cancer cells (HT-29) and facilitated with an LC/MS dereplication procedure. The structures of the new compounds (1-8) were determined on the basis of spectroscopic data interpretation. The Mosher ester method was employed to determine the absolute configurations of 5-7, and the absolute configuration of the 9,10-diol unit of compound 8 was established by a dimolybdenum tetraacetate [Mo2(AcO)4] induced circular dichroism procedure. Seven known rocaglate derivatives (9-15) exhibited significant cytotoxicity against the HT-29 cell line, with rocaglaol (9) being the most potent (ED50 0.0007 µM). The new compounds 2-4 were also active against this cell line, with ED50 values ranging from 0.46 to 4.7 µM. The cytotoxic compounds were evaluated against a normal colon cell line, CCD-112CoN. In addition, the new compound perviridicin B (2), three known rocaglate derivatives (9, 11, 12), and a known sesquiterpene, 2-oxaisodauc-5-en-12-al (17), showed significant NF-κB (p65) inhibitory activity in an ELISA assay.

Cytotoxic and NF-κB inhibitory sesquiterpene lactones from Piptocoma rufescens.

Six new (1-6) and eight known germacranolide-type sesquiterpene lactones, along with several known phenylpropanol coumarates and methylated flavonoids, were isolated from the leaves of Piptocoma rufescens, collected in the Dominican Republic. The new compounds were identified by analysis of their spectroscopic data, with the molecular structure of 3 being established by single-crystal X-ray diffraction. The absolute configurations of the sesquiterpene lactones isolated were determined from their CD and NOESY NMR spectra, together with the analysis of Mosher ester reactions. Bioassay screening results showed the majority of the sesquiterpene lactones isolated (1-13) to be highly cytotoxic toward the HT-29 human colon cancer cell line, with the most potent compound being 15-deoxygoyazensolide (10, IC(50), 0.26 µM). In addition, several of the sesquiterpene lactones exhibited NF-κB (p65) inhibitory activity.

Secondary metabolites from Garcinia daedalanthera Pierre leaves (Clusiaceae).

Two new glycerol esters, (S)-2-hydroxy-3-(octanoyloxy)propyl tetracosanoate (1) and (S)-3-(((S)-11-acetoxy octadecanoyl)oxy)propane-1,2-diyl diacetate (2), and eight known compounds, docosanedioic acid (3), 2,5-dimethylnonadecane (4), lupeol (5), stigmasterol (6), ß-sitosterol (7), heptadecanoic acid (8), hexanedioic acid, 1,6-bis[(2R)-ethylhexyl] ester (9), and 1,3-di-O-[2',2'-di-(p-phenylene)] (10) were isolated from the leaves of Garcinia daedalanthera Pierre, collected from Indonesia. Structural analysis of the isolates was performed using 1 D- and 2 D-NMR, LC- and GC-MS, IR, polarimetry, and UV-visible spectroscopic methods. Cytotoxicity assessments, as well as reactive oxygen species (ROS) analysis of the isolates, were also completed. Lupeol was the only compound found active with an IC50 value of 19.2 µM against HT-29 colon cancer cells. Significant ROS inhibition and induction activity was observed for compounds 4 and 8, respectively.

Activity in MCF-7 Estrogen-sensitive Breast Cancer Cells of Capsicodendrin from Cinnamosma fragrans.

BACKGROUND/AIM: Effect of capsicodendrin on the NF-κB pathway was studied in MCF-7 cancer cells. MATERIALS AND METHODS: The transcription factor assay was used to screen for NF-κB activity. The effect on IKKß, ICAM-1, and caspase-7 were studied using western blot. Caspase-1 was studied using Promega Caspase-Glo® assay. Reactive oxygen species (ROS) were detected using the fluorescent probe DCFH-DA. The potentiometric dye JC-1 was used to assess mitochondrial membrane potential (ΔΨm) and the cell cycle was examined using a fluorescence-activated cell sorter. RESULTS: NF-κB p65 inhibitory effect was IC50=8.6 µM and cytotoxic activity was IC50=7.5 µM. The upstream IKK and the downstream ICAM-1 were down-regulated. Sub G1-phase population increased to 81% after 12 h of treatment with capsicodendrin (10 µM) and there was no loss of ΔΨM. CONCLUSION: Increased levels of intracellular ROS promoted activity of caspase-1 and induced cell death in MCF-7 cells. Capsicodendrin may be a future anticancer agent that prevents the progression of metastatic breast cancer.

Benzophenones and biflavonoids from Rheedia edulis.

Two new polyisoprenylated benzophenones, 32-hydroxy-ent-guttiferone M (1) and 6-epi-guttiferone J (2), along with seven known compounds, 6-epi-clusianone (3), guttiferone A (4), xanthochymol (5), guttiferone E (6), isoxanthochymol (7), (+)-volkensiflavone (8), and (+)-morelloflavone (9), were identified from the seeds and rinds of Rheedia edulis. Compounds 1-3 and 5-9 have been isolated and identified from this species for the first time. The structures of the new compounds were elucidated mainly by analysis of their 1D and 2D NMR spectroscopic data, and their absolute configurations were determined by comparison of their experimental optical rotation and electronic circular dichroism measurements with those values predicted by DFT calculations. Compound 1 showed significant antioxidant activity in both DPPH and ABTS free radical scavenging assays, whereas compound 2 was inactive.

Novel narrow spectrum benzyl thiophene sulfonamide derivatives to control Campylobacter.

Campylobacter is a leading cause of bacterial foodborne gastroenteritis worldwide, and poultry are a major source of human campylobacteriosis. The control of Campylobacter from farm to fork is challenging due to emergence of microbial resistance and lack of effective control methods. We identified a benzyl thiophene sulfonamide based small molecule (compound 1) with a minimal inhibitory concentration (MIC) of 100 µM against Campylobacter jejuni 81-176 and Campylobacter coli ATCC33559, good drug-like properties, and low toxicity on eukaryotic cells. Compound 1 was used as a lead for the preparation of 13 analogues. Two analogues, compounds 4 and 8 (TH-4 and TH-8), were identified with better antimicrobial properties than compound 1. TH-4 and TH-8 had a MIC of 12.5 µM and 25 µM for C. coli and 50 µM and 100 µM for C. jejuni, respectively. Cytological studies revealed that both compounds affected C. jejuni envelope integrity. Further, both compounds had no effect on other foodborne pathogens. TH-4 and TH-8 had a minimal impact on the chicken cecal microbiota and were not toxic to colon epithelial cells and chicken macrophages, and red blood cells at 200 µM. Further, TH-4 and TH-8 reduced the Campylobacter load in chicken ceca (up to 2-log reduction) when infected chickens were orally treated for 5 days with 0.254 mg kg-1; as well as against internalized Campylobacter in Caco-2 cells at 12.5 µM and higher. Our study identified two novel specific and safe benzyl thiophene sulfonamide derivatives having potential for control of Campylobacter in chickens and humans.

Hapalindole H Induces Apoptosis as an Inhibitor of NF-ĸB and Affects the Intrinsic Mitochondrial Pathway in PC-3 Androgen-insensitive Prostate Cancer Cells.

BACKGROUND: Prostate cancer presents the highest incidence rates among all cancers in men. Hapalindole H (Hap H), isolated from Fischerella muscicola (UTEX strain number LB1829) as part of our natural product anticancer drug discovery program, was found to be significantly active against prostate cancer cells. MATERIALS AND METHODS: In this study, Hap H was tested for nuclear factor-kappa B (NF-ĸB) inhibition and selective cytotoxic activity against different cancer cell lines. The apoptotic effect was assessed on PC-3 prostate cancer cells by fluorescence-activated cell sorting analysis. The underlying mechanism that induced apoptosis was studied and the effect of Hap H on mitochondria was evaluated and characterized using western blot and flow cytometric analysis. RESULTS: Hap H was identified as a potent NF-ĸB inhibitor (0.76 µM) with selective cytotoxicity against the PC-3 prostate cancer cell line (0.02 µM). The apoptotic effect was studied on PC-3 cells. The results showed that treatment of PC-3 cells with Hap H reduced the formation of NAD(P)H, suggesting that the function of the outer mitochondrial membrane was negatively affected. Thus, the mitochondrial transmembrane potential was assessed in Hap H treated cells. The results showed that the outer mitochondrial membrane was disrupted as an increased amount of JC-1 monomers were detected in treated cells (78.3%) when compared to untreated cells (10.1%), also suggesting that a large number of treated cells went into an apoptotic state. CONCLUSION: Hap H was found to have potent NF-ĸB p65-inhibitory activity and induced apoptosis through the intrinsic mitochondrial pathway in hormone-independent PC-3 prostate cancer cells.

Structure-Activity Relationship of Niclosamide Derivatives.

BACKGROUND/AIM: Cancer is a leading cause of death. Hence, this study aimed at the optimization of niclosamide derivatives for the development of new potential anticancer agents. MATERIALS AND METHODS: Niclosamide derivatives were synthesized and tested against a panel of human cancer cells: MDA and MCF7 breast cancer cells, PC3 and DU-145 prostate cancer cells, Hela cervical cancer cells, and HL-60 acute promyelocytic leukemia cells. They were also tested in nuclear factor-ĸappa B (NFĸB), V-Ki-ras2 Kirsten rat sarcoma viral oncogene (KRAS), and mitochondria transmembrane potential (MTP) assays. RESULTS: N-(3,5-Bis(trifluoromethyl)phenyl)-5-chloro-2-hydroxybenzamide exhibited the most significant cytotoxicity against HL-60 cells, while 5-chloro-N-(2-chlorophenyl)-2-hydroxybenzamide was the most active in the NFĸB assay and 5-chloro-N-(3,5-difluorophenyl)-2-hydroxybenzamide in the MTP assay. 5-chloro-N-(2-chloro-4-(trifluoromethyl) phenyl)-2-hydroxybenzamide and 5-chloro-2-hydroxy-N-(4-hydroxyphenyl)benzamide inhibited both HL-60 cell proliferation and NFĸB. CONCLUSION: In-depth study of the most promising compounds is highly encouraged to further develop into potential anticancer agents those derivatives found to be significantly active.

Differential Effect of Wortmannolone Derivatives on MDA-MB-231 Breast Cancer Cells.

BACKGROUND/AIM: The survival rate of women diagnosed with triple-negative breast-cancer (TNBC) remains low. Hence, this study aimed at the chemical and biological optimization of furanosteroid derivatives for the treatment of this type of malignancy using TNBC cells. MATERIALS AND METHODS: Semi-synthetic analogs of wortmannolone (1-6) that negatively affected the aberrant pathways in tumor cells were evaluated in hormone-independent breast cancer cells using western blot and cell-cycle analysis. RESULTS: Wortmannolone derivatization generated NF-ĸB inhibitors as new lead structures for further development. Compound (3) was found to be the most significantly active lead. CONCLUSION: Structure-activity analysis in the present study showed that acetylation of the hydroxyl groups and substitution on C3 and C17 of wortmannolone enhanced biological activity. Alpha-substitution of the acetyl group in C3 on ring A (compound 3) resulted in ROS inducing effect; however, presence of an acetyl group in ß-position of C3 displayed the highest NF-ĸB p65 inhibitory activity (0.60 µM).

Ambiguine I Isonitrile from Fischerella ambigua Induces Caspase-Independent Cell Death in MCF-7 Hormone Dependent Breast Cancer Cells.

Ambiguine I isonitrile (AmbI) obtained from the cultured cyanobacterium Fischerella ambigua was identified as a potent NF-κB inhibitor (IC50=30 nM). The cytotoxic effect was evaluated in both HT-29 colon cancer cell line (EC50=4.35 µM) and MCF-7 breast cancer cell line (EC50=1.7 µM) using the SRB assay. In the cells treated with AmbI, an increased population of cells was detected in sub G1-phase. The apoptotic effect was associated with block in G1-phase of the cell cycle in treated cells; however, cell death was induced independently of caspase-7. The NF-κB expression of p50 and p65 units were also examined in treated cells and compared with the positive control, rocaglamide (IC50=75 nM). Moreover, the expression of mediators of the NF-κB pathway such as kinase IKKκ was studied at increasing concentrations of AmbI. The down stream effect of NF-κB inhibition and the effect on the expression of TNF-α induced ICAM-1 was evaluated. Thus, the dose-dependent and time-dependent effect of AmbI on MCF-7 cells was examined in an attempt to investigate its potential mechanism of action on inducing apoptosis.

Apoptotic Effect of Wortmannolone on Cancer Cells through Potent ROS Induction.

Nuclear factor κappa-B inhibitors isolated from natural sources that induce apoptosis are promising new agents with anticancer properties. Wortmannin and wortmannolone were isolated from endophytic fungus (Penicillum polonicum) and showed NF-κB inhibitory effects with inhibitory concentration (IC50) of 0.47 µM and 2.06 µM for wortmannin and wortmannolone, respectively. The activity was compared with rocaglamide (IC50=0.075 µM). The mechanism through which wortmannin and wortmannolone exhibited an attenuating effect on the NF-κB pathway was further evaluated in this study. Wortmannolone showed significant reactive oxygen species (ROS) inducing effects in HeLa cervical cells. The ROS inducing effect was concentration dependent, and the ROS generating activity was comparable with daunomycin, a potent chemotherapeutic agent. The findings suggested that the elevated formation of ROS was partially involved in the induction of apoptosis in treated cells. Potent cytotoxic and apoptotic effects were also displayed in MDA-MB-231 hormone independent breast cancer cells when treated with wortmannolone (IC50=3.79 nM). Thus, wortmannolone, a furanosteroid from an endophytic fungus, is a promising agent for further drug development.

Garcinia benzophenones inhibit the growth of human colon cancer cells and synergize with sulindac sulfide and turmeric.

Previous studies indicate that extracts and purified components from Garcinia species inhibit the growth of human colon cancer cells. Garcinia benzophenones activate the expression of genes in the endoplasmic reticulum and cellular energy stress (mTOR) pathways. This study examines the growth inhibitory and synergistic effects of Garcinia benzophenones, alone or combined with chemopreventive agents, on human colon cancer cells. To find optimal combination treatments, HT29 colon cancer cells were treated with benzophenones alone, or combined with chemopreventive agents, and cell growth measured using the MTT assay. To reveal effects on signaling pathways, we assessed effects of the MEK inhibitor U0126 and the ER IP3 receptor antagonist heparin, as well as effects on the phosphorylation of 4E-BP-1 (mTOR pathway), using Western blot analysis. New and known benzophenones from Garcinia intermedia inhibited the growth of human colon cancer cells; an alcohol extract of Garcinia xanthochymus, as well as purified guttiferones (guttiferone E and xanthochymol), preferentially inhibited the growth of colon cancer versus nonmalignant intestinal epithelial cells. Guttiferone E exhibited synergy with the NSAID sulindac sulfide and xanthochymol, with the spice turmeric. Guttiferone A did not alter phosphorylation of 4E-BP-1, indicating that the mTORC1 pathway is not involved in its action. The effects of xanthochymol were enhanced by U0126, at low doses, and were blocked by heparin, indicating that the MEK pathway is involved, while the ER IP3 receptor is critical for its action. These studies indicate the potential of benzophenones, alone or combined with sulindac sulfide or turmeric, to prevent and treat colon cancer.

Goyazensolide Induces Apoptosis in Cancer Cells in vitro and in vivo.

As part of the screening program for anticancer agents from natural sources, the sesquiterpene lactone goyazensolide (GZL) was identified as a potent NF-κB inhibitor. The hollow-fiber assay was used to evaluate the anti-tumor efficacy of GZL in vivo. The mechanistic effects of GZL were evaluated in the HT-29 colonic cell line to reveal the pathway through which GZL exerts its effects. NF-κB subunits p65 and p50 were inhibited, and the upstream mediator IκB kinase (IKKß) was downregulated in a dose-dependent manner. Apoptosis was mediated by caspase-3, and cell cycle arrest was detected in G1-phase. Consequently, 96% of the cell population was in sub G1-phase after treatment with GZL (10 µM).The antitumor effect of GZL was observed at a dose of 12.5 mg/kg. Cell adhesion was affected as a result of NF-κB inhibition. GZL appears to selectively target the transcription factor NF-κB. In summary, GZL sensitizes HT-29 colon cancer cells to apoptosis and cell death in a dose-dependent manner both in vivo and in vitro, through NF-κB inhibition (IC50 = 3.8 µM). Thus, it is a new potent lead compound for further development into a new effective chemotherapeutic agent.

Effects of (5Z)-7-oxozeaenol on MDA-MB-231 breast cancer cells.

AIM: (5Z)-7-Oxozeaenol was studied to reveal the path through which it exerts its effects on triple-negative MDA-MB-231 breast cancer cells. MATERIALS AND METHODS: The apoptotic effect of (5Z)-7-oxozeaenol on MDA-MB-231 cancer cells was analyzed by cell flow cytometry. The effects of (5Z)-7-oxozeaenol on the expression of the nuclear factor kappa B (NF-κB) p65, p50, IκB kinase (IKKα), IKKß and caspase-7 were analyzed by western blot. The expression of intracellular reactive oxygen species (ROS) and effects on cell adhesion were also assessed. Cell viability was determined using the 3[4,5-dimethylthiazol-2-yl-]2,5-diphenyl tetrazolium bromide (MTT) assay. RESULTS: (5Z)-7-Oxozeaenol down-regulated NF-κB in a dose-dependent manner. Intracellular levels of ROS increased in a dose-dependent manner when treated with (5Z)-7-oxozeaenol and potentiated in the presence of H(2)O(2), when compared to paclitaxel which was used as positive control. Treatment with (5Z)-7-oxozeaenol resulted in G1-phase arrest of treated cells and inhibition of cell proliferation. Cell adhesion was notably affected in treated cells. (5Z)-7-Oxozeaenol also significantly enhanced apoptosis of treated cells, through the activation of caspase-7. CONCLUSION: Our findings suggest that (5Z)-7-oxozeaenol is a potent up-stream inhibitor of the NF-κB pathway, enhances the sensitivity of treated cells to apoptosis induced by ROS, and affects cell adhesion of MDA-MB-231 breast cancer cells. Thus, (5Z)-7-oxozeaenol is a potential new lead for breast cancer drug development since it might, in combination therapy, enhance the efficacy of current treatments and reduce resistance to chemotherapy of triple negative breast cancer.

Effects of (5Z)-7-oxozeaenol on the oxidative pathway of cancer cells.

AIM: As part of an on going investigation of novel anticancer agents from natural origin, the biological and cellular effects of (5Z)-7-oxozeaenol on cancer cells were investigated. MATERIALS AND METHODS: The expression of nuclear factor kappa B (NF-κB), IκB kinase (IKKα), IKKß and caspase-3 were analyzed by western blot. Reactive oxygen species (ROS) fluorescence and caspase luminescent assays were used to assess the intracellular effects in HeLa cervical and HT-29 colon cancer cell lines. The mitochondrial transmembrane potential (MTP) was analyzed by fluorescence-activated cell sorting (FACS). RESULTS: Cells treated with (5Z)-7-oxozeaenol exhibited down-regulation of NF-κB in a dose-dependent manner. Treatment with (5Z)-7-oxozeaenol significantly enhanced the levels of ROS in HeLa and HT-29 cells. MTP was reduced in HT-29 cells. The expression of caspase-3 and -7 was induced in (5Z)-7-oxozeaenol treated HeLa cells, in comparison with those treated with paclitaxel. CONCLUSION: Our findings suggest that (5Z)-7-oxozeaenol is a potent inhibitor of the NF-κB pathway and potentiates the production of ROS, as well as induces caspase-3 and -7 in HeLa and HT-29 cancer cells. Thus, (5Z)-7-oxozeaenol represents a new lead compound for drug development, particularly as a new cancer chemotherapeutic agent, since programmed cell death might be mediated through the activation of a caspase-arbitrated pathway.

Polyisoprenylated benzophenones from Clusiaceae: potential drugs and lead compounds.

Many new polyisoprenylated benzophenones with a bicyclo[3.3.1]-nonane-2,4,9-trione core structure have been isolated from plants in the Clusiaceae family, and their potent biological properties have been the subject of several studies. This review summarizes the biological activities reported for these secondary metabolites including cytotoxic, antimicrobial, antioxidant, and anti-inflammatory activities. Our efforts during the past years have foremost been directed towards isolating new polyisoprenylated benzophenones, as well as understanding the possible target and mechanism of action through which these compounds arrest cancer cells and inhibit the progression of the cell-cycle. The transcription of genes is affected in cancer cells treated with polyisoprenylated benzophenones; the oncogene c-Myb is down-regulated and endoplasmatic stress genes XBP1, ATF4, and DDIT3/CHOP are turned on. Consequently, the expression of iNOS and cell cycle regulators such as cyclin D and E are reduced. Evidence presented by independent investigators suggests that polyisoprenylated benzophenones affect the mediators in the Akt/mTOR stress pathway, although the specific target remains to be discovered. In addition, benzophenones isolated from plants display high antioxidant capacity and protect cells from oxidative stress and the formation of ROS involved during the inflammatory process. Since antiviral activity was initially reported for guttiferone A, potent synthetic analogues have been developed as effective new non-nucleoside reverse transcriptase inhibitors (NNRTI) to treat drug resistant HIV-1. In addition, benzophenones exert antimicrobial effects particularly against MRSA. The structure-activity relationships of polyisoprenylated benzophenones from natural sources and those of synthetic analogues are included in this review. Absorption, metabolism, and elimination of benzophenones are also discussed.

Antioxidant capacities of ten edible North American plants.

The EtOAc extract obtained from ten edible North American plants, Acorus calamus, Clintonia borealis, Gaultheria shallon, Juniperus osteosperma, Opuntia polyacantha, Prunus americana, Prunus virginiana, Sambucus cerulea, Sorbus americana and Vaccinium parvifolium, were tested in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical assay. High antioxidant activity was obtained from the extracts of three fruits, Gaultheria shallon, Sambucus cerulea and Prunus americana and one extracted rhizome, Acorus calamus. Catechin and epicatechin, potent polyphenolic antioxidants, were identified in the EtOAc extracts of Gaultheria shallon and Sambucus cerulea by reversed-phase thin-layer chromatography (TLC) and reversed-phase high-performance liquid chromatography (HPLC).