The Marine Natural Compound Dragmacidin D Selectively Induces Apoptosis in Triple-Negative Breast Cancer Spheroids.

Cancer cells grown in 3D spheroid cultures are considered more predictive for clinical efficacy. The marine natural product dragmacidin D induces apoptosis in MDA-MB-231 and MDA-MB-468 triple-negative breast cancer (TNBC) spheroids within 24 h of treatment while showing no cytotoxicity against the same cells grown in monolayers and treated for 72 h. The IC50 for cytotoxicity based on caspase 3/7 cleavage in the spheroid assay was 8 ± 1 µM in MDA-MB-231 cells and 16 ± 0.6 µM in MDA-MB-468 cells at 24 h. No cytotoxicity was seen at all in 2D, even at the highest concentration tested. Thus, the IC50 for cytotoxicity in the MTT assay (2D) in these cells was found to be >75 µM at 72 h. Dragmacidin D exhibited synergy when used in conjunction with paclitaxel, a current treatment for TNBC. Studies into the signaling changes using a reverse-phase protein array showed that treatment with dragmacidin D caused significant decreases in histones. Differential protein expression was used to hypothesize that its potential mechanism of action involves acting as a protein synthesis inhibitor or a ribonucleotide reductase inhibitor. Further testing is necessary to validate this hypothesis. Dragmacidin D also caused a slight decrease in an invasion assay in the MDA-MB-231 cells, although this failed to be statistically significant. Dragmacidin D shows intriguing selectivity for spheroids and has the potential to be a treatment option for triple-negative breast cancer, which merits further research into understanding this activity.

High-Throughput Screening of a Marine Compound Library Identifies Anti-Cryptosporidium Activity of Leiodolide A.

Cryptosporidium sp. are apicomplexan parasites that cause significant morbidity and possible mortality in humans and valuable livestock. There are no drugs on the market that are effective in the population most severely affected by this parasite. This study is the first high-throughput screen for potent anti-Cryptosporidium natural products sourced from a unique marine compound library. The Harbor Branch Oceanographic Institute at Florida Atlantic University has a collection of diverse marine organisms some of which have been subjected to medium pressure liquid chromatography to create an enriched fraction library. Numerous active compounds have been discovered from this library, but it has not been tested against Cryptosporidium parvum. A high-throughput in vitro growth inhibition assay was used to test 3764 fractions in the library, leading to the identification of 23 fractions that potently inhibited the growth of Cryptosporidium parvum. Bioassay guided fractionation of active fractions from a deep-sea sponge, Leiodermatium sp., resulted in the purification of leiodolide A, the major active compound in the organism. Leiodolide A displayed specific anti-Cryptosporidium activity at a half maximal effective concentration of 103.5 nM with selectivity indexes (SI) of 45.1, 11.9, 19.6 and 14.3 for human ileocecal colorectal adenocarcinoma cells (HCT-8), human hepatocellular carcinoma cells (Hep G2), human neuroblastoma cells (SH-SY5Y) and green monkey kidney cells (Vero), respectively. The unique structure of leiodolide A provides a valuable drug scaffold on which to develop new anti-Cryptosporidium compounds and supports the importance of screening natural product libraries for new chemical scaffolds.

The Marine Natural Product Furospinulosin 1 Induces Apoptosis in MDA-MB-231 Triple Negative Breast Cancer Cell Spheroids, But Not in Cells Grown Traditionally with Longer Treatment.

Cancer cells grown in spheroid conditions interact with each other and the extracellular matrix, providing a better representation of the in vivo environment than two-dimensional cultures and are a more clinically relevant model. A discrete screening of genetically diverse marine samples in the spheroid assay led to the identification of a novel activity for the known compound furospinulosin 1. This compound shows activity against MDA-MB-231 triple negative breast cancer cells grown as spheroids and treated for 24 or 48 h. No cytotoxicity was seen in traditional two-dimensional adherent cultures treated for a longer time (72 h). A reverse phase protein array (RPPA) confirmed the limited activity of the compound in cells grown traditionally and revealed changes in protein expression when cells are grown as spheroids that are associated with better clinical prognosis. Analysis of the RPPA data through the Broad institute's connectivity map suggested the hypothesis that furospinulosin 1 functions as an MEK inhibitor. Analysis of the RPPA data through STRING supports the apoptosis observed. The selectivity exhibited by furospinulosin 1 for triple negative breast cancer cells only when grown as spheroids makes it an interesting compound with strong therapeutic potential that merits further study.

Discovery of Survivin Inhibitors Part 1: Screening the Harbor Branch Pure Compound Library.

Survivin is a 16.5 KDa protein whose functions include promoting cellular mitosis, angiogenesis, and senescence as well as inhibiting apoptosis. Higher survivin expression is found in cancer tissues than normal tissues, and this expression correlates with disease progression and aggressiveness. Survivin has been validated as a clinical target for cancer. Small molecules are important antagonists of survivin levels in cancer cells. A structurally diverse library of genetically encoded small molecules (natural products) derived from marine plants, invertebrates, and microbes was screened for their ability to reduce expression levels of survivin in the DLD-1 colon adenocarcinoma and the A549 nonsmall cell lung carcinoma cell lines. This led to the identification of this novel activity for the known compounds eryloside E, ilicicolin H, tanzawaic acid A, and p-hydroxyphenopyrrozin. Both eryloside E and ilicicolin H showed the ability to reduce survivin expression in the low micromolar range against both cell lines.

Dragmacidin G, a Bioactive Bis-Indole Alkaloid from a Deep-Water Sponge of the Genus Spongosorites.

A deep-water sponge of the genus Spongosorites has yielded a bis-indole alkaloid which we have named dragmacidin G. Dragmacidin G was first reported by us in the patent literature and has recently been reported by Hitora et al. from a sponge of the genus Lipastrotheya. Dragmacidin G is the first in this series of compounds to have a pyrazine ring linking the two indole rings. It also has a rare N-(2-mercaptoethyl)-guanidine side chain. Dragmacidin G shows a broad spectrum of biological activity including inhibition of methicillin-resistant Staphylococcus aureus, Mycobacterium tuberculosis, Plasmodium falciparum, and a panel of pancreatic cancer cell lines.

Analogues of the Potent Antitumor Compound Leiodermatolide from a Deep-Water Sponge of the Genus Leiodermatium.

Two new analogues of the potent antitumor compound leiodermatolide, which we call leiodermatolides B and C, have been isolated from specimens of a deep-water sponge of the genus Leiodermatium collected off Florida. The compounds were purified using standard chromatographic methods, and the structures defined through interpretation of the HRMS and 1D and 2D NMR data. Leiodermatolide B (2) lacks the C-21 hydroxy group found in leiodermatolide and has equal potency as the parent compound, providing a simpler analogue for possible clinical development. It inhibits the proliferation of the AsPC-1 human pancreatic adenocarcinoma cell line with an IC50 of 43 nM. Leiodermatolide C (3) has a modified macrolide ring and is over 85-fold less potent with an IC50 of 3.7 µM against the same cell line. These compounds add to the knowledge of the pharmacophore of this class of potent antitumor agents.

Inhibition of IL-8 secretion on BxPC-3 and MIA PaCa-2 cells and induction of cytotoxicity in pancreatic cancer cells with marine natural products.

Pancreatic cancer presents one of the most negative prognosis of all cancers as it has usually metastasized by the time a patient is diagnosed. The American Cancer Society estimates that 93% of patients will die within 5 years of diagnosis, highlighting the need for new drugs to treat this disease. Interleukin 8 (IL-8) mediates the angiogenesis of tumors arising from Ras mutations, which are present in about 90% of pancreatic adenocarcinomas. Overexpression of IL-8 in pancreatic tumors is believed to promote tumor angiogenesis and to activate survival signaling pathways. A 96-well cell-based enzyme-linked immunosorbent assay was set up to screen the Harbor Branch Oceanographic Institute library of marine natural products to identify those with the ability to inhibit IL-8 production by BxPC-3 pancreatic cancer cells. Over 1000 fractions were screened, resulting in the identification of 10 known marine natural products with this ability. These compounds fall into four classes of compounds including the pyrroloiminoquinone alkaloids secobatzelline A and isobatzelline C; mycalamide A and B, onnamide A, discalamide A, and theopederin K from the mycalamide class of polyketides; the lipopeptide microcolin A; and the cyclic depsipeptides didemnin B and nordidemnin B. In addition, didemnin B, nordidemnin B, and theopederin K induce potent cytotoxicity against four pancreatic cancer cell lines tested. Many of these compounds have been previously reported to inhibit protein synthesis and the decrease in IL-8 production may be nonspecific. Nevertheless, this is a new activity for these compounds and inhibition of IL-8 secretion by pancreatic cancer cells can now be added to the previously reported antiangiogenic activities of the didemnins.

Leiodermatolide, a novel marine natural product, has potent cytotoxic and antimitotic activity against cancer cells, appears to affect microtubule dynamics, and exhibits antitumor activity.

Pancreatic cancer, the fourth leading cause of cancer death in the United States, has a negative prognosis because metastasis occurs before symptoms manifest. Leiodermatolide, a polyketide macrolide with antimitotic activity isolated from a deep water sponge of the genus Leiodermatium, exhibits potent and selective cytotoxicity toward the pancreatic cancer cell lines AsPC-1, PANC-1, BxPC-3, and MIA PaCa-2, and potent cytotoxicity against skin, breast and colon cancer cell lines. Induction of apoptosis by leiodermatolide was confirmed in the AsPC-1, BxPC-3 and MIA PaCa-2 cells. Leiodermatolide induces cell cycle arrest but has no effects on in vitro polymerization or depolymerization of tubulin alone, while it enhances polymerization of tubulin containing microtubule associated proteins (MAPs). Observations through confocal microscopy show that leiodermatolide, at low concentrations, causes minimal effects on polymerization or depolymerization of the microtubule network in interphase cells, but disruption of spindle formation in mitotic cells. At higher concentrations, depolymerization of the microtubule network is observed. Visualization of the growing microtubule in HeLa cells expressing GFP-tagged plus end binding protein EB-1 showed that leiodermatolide stopped the polymerization of tubulin. These results suggest that leiodermatolide may affect tubulin dynamics without directly interacting with tubulin and hint at a unique mechanism of action. In a mouse model of metastatic pancreatic cancer, leiodermatolide exhibited significant tumor reduction when compared to gemcitabine and controls. The antitumor activities of leiodermatolide, as well as the proven utility of antimitotic compounds against cancer, make leiodermatolide an interesting compound with potential chemotherapeutic effects that may merit further research.

Toward the identification, characterization and experimental culture of Lacazia loboi from Atlantic bottlenose dolphin (Tursiops truncatus).

Lobomycosis (lacaziosis) is a chronic, granulomatous, fungal infection of the skin and subcutaneous tissues of humans and dolphins. To date, the causative agent, the yeast-like organism Lacazia loboi, has not been grown in the laboratory, and there have been no recent reports describing attempts to culture the organism. As a result, studies on the efficacy of therapeutics and potential environmental reservoirs have not been conducted. Therefore, the objective of the current study was to utilize both classical and novel microbiological methods in order to stimulate growth of Lacazia cells collected from dolphin lesions. This included the experimental inoculation of novel media, cell culture, and the use of artificial skin matrices. Although unsuccessful, the methods and results of this study provide important insight into new approaches that could be utilized in future investigations of this elusive organism.

The marine natural product microsclerodermin A is a novel inhibitor of the nuclear factor kappa B and induces apoptosis in pancreatic cancer cells.

Pancreatic cancer, the 4th leading cause of cancer death in the US, is highly resistant to all current chemotherapies, and its growth is facilitated by chronic inflammation. An important mediator of inflammation is the nuclear factor kappa B (NFκB), a transcription factor that regulates over 500 genes including the regulation of anti-apoptotic proteins, cell cycle progression and cytokine production. NFκB is constitutively activated in pancreatic cancer cells contributing to their resistance to apoptosis and high metastatic potential. Although many small molecules that inhibit NFκB have been identified, none are currently used in the clinic, perhaps due to their lack of specificity. To identify novel inhibitors of NFκB, the HBOI library of enriched fractions from marine organisms was screened using a reporter cell line that produces luciferin under the transcriptional control of NFκB. Fractions from the sponge Amphibleptula were active in this screen and contained the antifungal cyclic peptide microsclerodermin A. Microsclerodermin A is shown here to inhibit NFκB transcriptional activity in a reporter cell line, to reduce levels of phosphorylated (active) NFκB in the AsPC-1 cell line, to have an IC50 for cytotoxicity in the low micromolar range against the AsPC-1, BxPC-3, MIA PaCa-2 and PANC-1 pancreatic cancer cell lines, and to induce significant apoptosis in the AsPC-1, BxPC-3 and the PANC-1 cell lines. Treatment of AsPC-1 cells with microsclerodermin A also resulted in an increase in IL-8 production without apparent induction of angiogenic factors and there is the possibility that inhibition of NFκB by microsclerodermin A is mediated by the glycogen synthase kinase 3ß pathway.

The marine natural product manzamine A targets vacuolar ATPases and inhibits autophagy in pancreatic cancer cells.

Manzamine A, a member of the manzamine alkaloids, was originally isolated from marine sponges of the genus Haliclona. It was recently shown to have activity against pancreatic cancer cells, but the precise mechanism of action remained unclear. To further our understanding of the mechanism of action of manzamine A, chemogenomic profiling in the yeast S. cerevisiae was performed, suggesting that manzamine A is an uncoupler of vacuolar ATPases. Fluorescence microscopy confirmed this effect on yeast vacuoles, where manzamine A produced a phenotype very similar to that of the established v-ATPase inhibitor bafilomycin A1. In pancreatic cancer cells, 10 µM manzamine A affected vacuolar ATPase activity and significantly increased the level of autophagosome marker LC3-II and p62/SQSTM1 as observed by western blot analysis. Treatment with manzamine A in combination with bafilomycin A1 (inhibitor of autophagosome-lysosome fusion) did not change the levels of LC3-II when compared to cells treated with bafilomycin A1 alone, suggesting that manzamine A is a potential inhibitor of autophagy by preventing autophagosome turnover. As autophagy is essential for pancreatic tumor growth, blocking this pathway with manzamine A suggests a promising strategy for the treatment of pancreatic cancer.

Neopetrosiquinones A and B, sesquiterpene benzoquinones isolated from the deep-water sponge Neopetrosia cf. proxima.

Two new marine-derived sesquiterpene benzoquinones which we designate as neopetrosiquinones A (1) and B (2), have been isolated from a deep-water sponge of the family Petrosiidae. The structures were elucidated on the basis of their spectroscopic data. Compounds 1 and 2 inhibit the in vitro proliferation of the DLD-1 human colorectal adenocarcinoma cell line with IC(50) values of 3.7 and 9.8 µM, respectively, and the PANC-1 human pancreatic carcinoma cell line with IC(50) values of 6.1 and 13.8 µM, respectively. Neopetrosiquinone A (1) also inhibited the in vitro proliferation of the AsPC-1 human pancreatic carcinoma cell line with an IC(50) value of 6.1 µM. The compounds are structurally related to alisiaquinone A, cyclozonarone, and xestoquinone.

A novel activity from an old compound: Manzamine A reduces the metastatic potential of AsPC-1 pancreatic cancer cells and sensitizes them to TRAIL-induced apoptosis.

PURPOSE: Pancreatic cancer is the fourth leading cause of cancer death in the United States, and new drugs to treat the disease are needed. Pancreatic cancer cells are highly metastatic and exhibit resistance to apoptosis. Small molecules that can restore sensitivity to apoptosis or reduce metastasis would have therapeutic potential against this disease. Manzamine A is an alkaloid isolated from marine sponges that was suspected to have inhibitory activity against the mitogen activated kinase kinase (MEK). Because of this, the effects of Manzamine A were studied in pancreatic cancer cells. METHODS: AsPC-1 cells were treated for 48 h in the presence of various concentrations of Manzamine A and their phenotype, cytotoxicity, cell invasion and susceptibility to apoptosis were observed. RESULTS: Manzamine A decreased single cell formation, abrogated cell migration and restored the susceptibility of the cells to TRAIL-induced apoptosis in AsPC-1 cells. Its mechanism of action remains unknown, as manzamine A does not inhibit MEK. CONCLUSIONS: Manzamine A appears to have a formerly unrecognized activity in blocking tumor cell invasion as well as in restoring cancer cell susceptibility to apoptosis in vitro and therefore has the potential to be used as an adjuvant to existing cancer therapies.

Early effects of lasonolide a on pancreatic cancer cells.

Lasonolide A, a novel polyketide-derived macrolide, was previously identified from an extract of the marine sponge Forcepia sp. in an assay for protein kinase C (PKC) inhibitors. Cytotoxicity testing and profiling of lasonolide A in the National Cancer Institute (NCI) 60 cell panel screen revealed that it was potent toward a broad range of cell lines and also suggested a unique mechanism of action. Contrary to expected results, we found lasonolide A to be a strong activator of PKC in Panc-1 pancreatic carcinoma cells. Downstream mitogen-activated protein kinases, ERK 1/2 and p38 were also rapidly phosphorylated in response to lasonolide A, as was Akt. Microscopy studies revealed that lasonolide A induced blebbing and contraction of the cells within minutes of exposure, and the eventual loss of adherence. However, membrane integrity was maintained and the effects were reversible if lasonolide A was washed from the cells after their loss of adherence. Pretreatment of cells with a myosin II inhibitor, blebbistatin, slowed the early onset, but did not prevent the morphological effects of lasonolide A. Cells stained for actin filaments showed some reduction in stress fiber structure after lasonolide A exposure; however, it did not affect the polymerization of purified actin in vitro. Bisindolemaleimide, a PKC inhibitor, and wortmannin, a phosphoinositide 3-kinase; inhibitor, did not reduce lasonolide A-induced contraction or blebbing or the activation of mitogen-activated protein kinases, although Akt phosphorylation was prevented by wortmannin pretreatment. Our results indicate that lasonolide A activates multiple signal transduction pathways and suggest that the origin is upstream of PKC.

Isolation, synthesis, and biological activity of aphrocallistin, an adenine-substituted bromotyramine metabolite from the Hexactinellida sponge Aphrocallistes beatrix.

A new adenine-substituted bromotyrosine-derived metabolite designated as aphrocallistin (1) has been isolated from the deep-water Hexactinellida sponge Aphrocallistes beatrix. Its structure was elucidated on the basis of spectral data and confirmed through a convergent, modular total synthetic route that is amenable toward future analogue preparation. Aphrocallistin inhibits the growth of a panel of human tumor cell lines with IC(50) values ranging from 7.5 to >100 microM and has been shown to induce G1 cell cycle arrest in the PANC-1 pancreatic carcinoma cell line. Aphrocallistin has been fully characterized in the NCI cancer cell line panel and has undergone in vitro ADME pharmacological profiling.

Mitf dosage as a primary determinant of melanocyte survival after ultraviolet irradiation.

Microphthalmia-associated transcription factor (Mitf) is essential for melanocyte development and function and regulates anti-apoptotic Bcl2 expression. We hypothesized that cellular deficiency of Mitf can influence melanocyte survival in response to ultraviolet (UV) radiation. Primary melanocyte cultures were prepared from neonatal wild-type mice and congenic animals heterozygous for Mitf mutations Mitf (mi-vga9/+) and Mitf(Mi-wh/+) and exposed to UV irradiation. Wild-type melanocytes were more resistant to UV-induced apoptosis than melanocytes partially deficient in Mitf activity, as determined by relative levels of intracellular melanin and relative activation of Mitf target genes Tyr, Tyrp1, Dct, and Cdk2. Comparative experiments with wild-type cells and congenic albino melanocytes demonstrated that these differences are not due to differences in melanin content, implicating Mitf as a primary determinant of UV-dependent melanocyte survival. Mitf activity correlated directly with resistance to UV-induced apoptosis in melanocytes. Mitf was important not only for regulating the expression of anti-apoptotic Bcl-2 following UV irradiation, but also the expression of the pro-apoptotic BH3-only Bad protein and activation of the extrinsic apoptotic pathway. Hence, Mitf is a multifaceted regulator of UV-induced apoptosis in melanocytes.

Selective cytotoxic activity of the marine-derived batzelline compounds against pancreatic cancer cell lines.

Pancreatic cancer is the fourth leading cause of cancer death in the United States. The prognosis of the disease is very negative, because the cancer will be usually metastasized by the time a patient manifests symptoms. Although combination therapy shows some promise, new drugs to treat the disease are needed. Given our interest in finding new therapies for pancreatic cancer, we sought to determine whether the known cytotoxic activity of the batzellines extended to pancreatic cancer cell lines. The batzellines are pyrroloiminoquinones alkaloids obtained from the deep-water Caribbean sponge Batzella sp (family Esperiopsidae, order Poecilosclerida). We show here that batzellines exhibit selective cytotoxicity towards the pancreatic cancer cell lines AsPC-1, Panc-1, BxPC-3, and MIA PaCa2 compared with the normal African green monkey kidney epithelial cell line Vero. The batzellines cause cytotoxicity by inducing cell cycle arrest that is mediated by their ability to intercalate into DNA and/or inhibit topoisomerase II activity. The cytotoxic abilities of isobatzellines A and C against pancreatic cancer cell lines, their low toxicity against normal cells, and their reported ability to be synthesized makes them interesting compounds with potential chemotherapeutic effects that may merit further research.

Local cytokine levels associated with delayed-type hypersensitivity responses: modulation by gender, ovariectomy, and estrogen replacement.

It is well established that females mount stronger immune responses than males, but only very little is understood about the underlying mechanisms. We have analyzed local cytokine differences among intact females, those that had been ovariectomized (OVX), those receiving estrogen replacement after OVX, and males, both before and after production of delayed-type hypersensitivity (DTH) responses. We report confirmation of a much larger DTH response in females versus males. However, OVX resulted in an even larger response, while estrogen replacement resulted in a smaller response when compared with intact females. In animals exposed for the first time to an antigen (without a DTH response), OVX increased interleukin-6 (IL-6) and estrogen replacement after OVX suppressed IL-6. Of the cytokines that differed between males and females exposed for the first time to an antigen, only IL-6 was higher in females versus males when exposure to antigen occurred for the second time (when the DTH response occurs). Analysis of cytokines with OVX and estrogen replacement after a second exposure to antigen showed that IL-6 did not significantly change. Levels of IL-4; Regulated upon Activation, Normal T-cell Expressed; and Secreted; and thrombopoietin, however, correlated with the DTH response, suggesting direct or indirect positive regulation by estrogen. These results suggest an important role for both IL-6 and IL-4 in determining the degree of DTH response, with IL-6 (which appears negatively regulated by estrogen) increasing and IL-4 (which appears positively regulated by estrogen) decreasing the response. The results further suggest that IL-6 may play a role in predisposing to a larger DTH response, while IL-4 levels seem more important during an active response.

Comparison of broadband UVB, narrowband UVB, broadband UVA and UVA1 on activation of apoptotic pathways in human peripheral blood mononuclear cells.

BACKGROUND/PURPOSE: Ultraviolet (UV) radiation is an important therapy for immune-mediated cutaneous diseases. Activation of early apoptotic pathways may play a role in the clinical effectiveness. Different UV wavelengths have different efficacy for various diseases, but it remains unclear whether the ability to induce apoptosis differs with respect to the wavelength, and whether they induce apoptosis through the same mechanism. The aim of this study is to analyze the effects of different UV wavelengths that are used clinically on normal human peripheral blood mononuclear cells (PBMCs). METHODS: PBMCs were treated with UV-light sources broadband UVB, narrowband UVB, broadband UVA and UVA1. Initiation of apoptosis was assessed by flow cytometry by staining-treated cells for activated caspases. Immunoblots were performed to measure for cleaved caspase-3, -8, -9, cytochrome c, Bcl 2-interacting domain and poly-(ADP ribose) polymerase cleavage. RESULTS: We demonstrate that all the UV radiation sources induced caspase activation in a dose-and time-dependent manner. Components of both the extrinsic and intrinsic pathways of apoptosis were activated by all of the UV wavelengths tested, but differed in the level of energy needed for activation. CONCLUSION: The greater effectiveness of UVB on initiation of apoptotic pathway suggests that apoptosis may play a role in the clinical efficacy of UVB-responsive inflammatory cutaneous diseases.