Extracts from Cladiella australis, Clavularia viridis and Klyxum simplex (Soft Corals) are Capable of Inhibiting the Growth of Human Oral Squamous Cell Carcinoma Cells

Many biomedical products have already been obtained from marine organisms. In order to search more therapeutic drugs against cancer, this study demonstrates the cytotoxicity effects of Cladiella australis, Clavularia viridis and Klyxum simplex extracts on human oral squamous cell carcinoma (SCC4, SCC9 and SCC25) cells using cell adhesion and cell viability assay. The morphological alterations in SCCs cells after treatment with three extracts, such as typical nuclear condensation, nuclear fragmentation and apoptotic bodies of cells were demonstrated by Hoechst stain. Flow cytometry indicated that three extracts sensitized SCC25 cells in the G₀/G₁ and S-G₂/M phases with a concomitant significantly increased sub-G₁ fraction, indicating cell death by apoptosis. This apoptosis process was accompanied by activation of caspase-3 expression after SCC25 cells were treated with three extracts. Thereby, it is possible that extracts of C. australis, C. viridis and K. simplex cause apoptosis of SCCs and warrant further research investigating the possible anti-oral cancer compounds in these soft corals.     

Cytotoxicity of the ascidian Cystodytes dellechiajei against tumor cells and study of the involvement of associated microbiota in the production of cytotoxic compounds

Many cytotoxic compounds of therapeutic interest have been isolated from marine invertebrates, and some of them have been reported to be of microbial origin. Pyridoacridine alkaloids are the main compounds extracted from the ascidian Cystodytes dellechiajei. Here we describe the in vitro antiproliferative activity against different tumor cell lines of the ascidian extracts and provide some insights on the role of the microbial community associated with the tunicate in the production of these compounds. C. dellechiajei extracts showed remarkably high antiproliferative activity (IC₅₀ ≤5 μg/mL) in human lung carcinoma A-549, colon adenocarcinoma H-116, pancreatic adenocarcinoma PSN-1 and breast carcinoma SKBR3 cell lines. Moreover, we found that the maximum activity was located in the tunic tissue of the colony, which harbours a microbial community. In order to ascertain the involvement of this community in the synthesis of the bioactive compounds different approachs that included culture and culture independent methods were carried out. We undertook a screening for antiproliferative activities of the bacterial isolates from the ascidian, as well as a comprative analysis of the cytotoxic activities and the microbial communities from two color morphs of the ascidian, green and blue. In addition, the changes of the antiproliferative activities and the composition of the microbial communities were studied from ascidians kept in aquaria and treated with antibiotics for one month. Our data obtained from the different experiments did not point out to bacteria as the source of the cytotoxic compounds, suggesting thus an ascidian origin.     

Purified Brominated Indole Derivatives from Dicathais orbita Induce Apoptosis and Cell Cycle Arrest in Colorectal Cancer Cell Lines

Dicathais orbita is a large Australian marine gastropod known to produce bioactive compounds with anticancer properties. In this research, we used bioassay guided fractionation from the egg mass extract of D. orbita using flash column chromatography and identified fractions containing tyrindoleninone and 6-bromoisatin as the most active against colon cancer cells HT29 and Caco-2. Liquid chromatography coupled with mass spectrometry (LCMS) and ¹H NMR were used to characterize the purity and chemical composition of the isolated compounds. An MTT assay was used to determine effects on cell viability. Necrosis and apoptosis induction using caspase/LDH assay and flow cytometry (PI/Annexin-V) and cell cycle analysis were also investigated. Our results show that semi-purified 6-bromoisatin had the highest anti-cancer activity by inhibiting cell viability (IC₅₀ = ~100 µM) and increasing caspase 3/7 activity in both of the cell lines at low concentration. The fraction containing 6-bromoisatin induced 77.6% apoptosis and arrested 25.7% of the cells in G2/M phase of cell cycle in HT29 cells. Tyrindoleninone was less potent but significantly decreased the viability of HT29 cells at IC₅₀ = 390 µM and induced apoptosis at 195 µM by increasing caspase 3/7 activity in these cells. This research will facilitate the development of these molluscan natural products as novel complementary medicines for colorectal cancer.     

Antitumor Effects of a Sesquiterpene Derivative from Marine Sponge in Human Breast Cancer Cells

In this study, the anti-proliferative effect of ilimaquinone, a sesquiterpene derivative from the marine sponge, in breast cancer cells was investigated. Ilimaquinone inhibited the proliferation of MCF-7 and MDA-MB-231 breast cancer cells with IC₅₀ values of 10.6 μM and 13.5 μM, respectively. Non-tumorigenic human breast epithelial cells were less sensitive to ilimaquinone than breast cancer cells. Flow cytometric and Western blot analysis showed that ilimaquinone induced S-phase arrest by modulating the expression of p-CDC-2 and p21. Ilimaquinone induces apoptosis, which is accompanied by multiple biological biomarkers, including the downregulation of Akt, ERK, and Bax, upregulation of p38, loss of mitochondrial membrane potential, increased reactive oxygen species generation, and induced autophagy. Collectively, these findings suggest that ilimaquinone causes cell cycle arrest as well as induces apoptosis and autophagy in breast cancer cells.     

Didemnaketals F and G,New Bioactive Spiroketals from a Red Sea Ascidian Didemnum Species

In continuation of our ongoing efforts to identify bioactive compounds from Red Sea marine organisms, a new collection of the ascidian Didemnum species was investigated. Chromatographic fractionation and HPLC purification of the CH₂Cl₂ fraction of an organic extract of the ascidian resulted in the identification of two new spiroketals, didemnaketals F (1) and G (2). The structure determination of the compounds was completed by extensive study of 1D (¹H, ¹³C, and DEPT) and 2D (COSY, HSQC, and HMBC) NMR experiments in addition to high-resolution mass spectral data. Didemnaketal F (1) and G (2) differ from the previously reported compounds of this class by the lack the terminal methyl ester at C-1 and the methyl functionality at C-2. Instead, 1 and 2 possess a methyl ketone moiety instead of the terminal ester. Furthermore, didemnaketal F possesses a disubstituted double bond between C-2 and C-3, while the double bond was replaced by a secondary alcohol at C-3 in didemnaketal G. In addition, they possess the unique spiroketal/hemiketal functionality which was previously reported in didemnaketal E. Didemnaketals F (1) and G (2) displayed moderate activity against HeLa cells with of IC_50s of 49.9 and 14.0 µM, respectively. In addition, didemnaketal F (1) displayed potent antimicrobial activity against E. coli and C. albicans. These findings provide further insight into the biosynthetic capabilities of this ascidian and the chemical diversity as well as the biological activity of this class of compounds.     

Identification and Bioactivity of Compounds from the Fungus Penicillium sp. CYE-87 Isolated from a Marine Tunicate

In the course of our continuous interest in identifying bioactive compounds from marine microbes, we have investigated a tunicate-derived fungus, Penicillium sp. CYE-87. A new compound with the 1,4-diazepane skeleton, terretrione D (2), together with the known compounds, methyl-2-([2-(1H-indol-3-yl)ethyl]carbamoyl)acetate (1), tryptamine (3), indole-3-carbaldehyde (4), 3,6-diisobutylpyrazin-2(1H)-one (5) and terretrione C (6), were isolated from Penicillium sp. CYE-87. The structures of the isolated compounds were established by spectral analysis, including 1D (¹H, ¹³C) and 2D (COSY, multiplicity edited-HSQC and HMBC) NMR and HRESIMS, as well as comparison of their NMR data with those in the literature. The compounds were evaluated for their antimigratory activity against the human breast cancer cell line (MDA-MB-231) and their antiproliferation activity against HeLa cells. Compounds 2 and 6 showed significant antimigratory activity against MDA-MB-231, as well as antifungal activity against C. albicans.     

Inhibitory Effects of Rosemary Extracts, Carnosic Acid and Rosmarinic Acid on the Growth of Various Human Cancer Cell Lines

The leaves of Rosmarinus officinalis harvested from three different locations of Turkey were extracted by both methanolic and supercritical CO₂ extraction. Subsequently, six extracts and the active compounds, carnosic acid, and rosmarinic acid were applied to various human cancer cell lines including NCI-H82 (human, small cell lung, carcinoma), DU-145 (human, prostate, carcinoma), Hep-3B (human, black, liver, carcinoma, hepatocellular), K-562 (human chronic myeloid leukemia), MCF-7 (human, breast, adenocarcinoma), PC-3 (human, prostate, adenocarcinoma) and MDA-MB-231 (human, breast, adenocarcinoma) by MTT assay. Supercritical CO₂ extracts had superior antiproliferative effect compared to the soxhlet extracts. Although the extracts exhibited various cytotoxic effects against different cell lines, comparatively low IC₅₀ values ranging between 12.50 and 47.55 μg/ml were attained against K-562, being the most sensitive cell line. Moreover, carnosic acid caused the lowest cell viability with values ranging from 13 to 30 % at a concentration of 19 μM after 48 h of treatments, resulting in superior antiproliferative effect. Rosemary extract is a potential candidate to be included in the anti-cancer diet with pre-determined doses avoiding toxicity.     

Oxygenated Acyclic Diterpenes with Anticancer Activity from the Irish Brown Seaweed Bifurcaria bifurcata

Brown alga Bifurcaria bifurcata is a prolific source of bioactive acyclic (linear) diterpenes with high structural diversity. In the continuation of our investigations on Irish brown algae, we undertook an in-depth chemical study on the n-hexanes and chloroform subextracts of B. bifurcata that led to isolation of six new (1–6) and two known (7–8) acyclic diterpenes. Chemical structures of the compounds were elucidated by a combination of 1D and 2D NMR, HRMS, FT-IR, [α]_D and vibrational circular dichroism (VCD) spectroscopy. Compounds 1–8, as well as three additional linear diterpenes (9–11), which we isolated from the same seaweed before, were tested against the human breast cancer cell line (MDA-MB-231). Several compounds moderately inhibited the growth of the MDA-MB-231 cell line with IC₅₀ values ranging from 11.6 to 32.0 μg/mL. The present study carried out on the lipophilic extracts of the Irish B. bifurcata shows the enormous capacity of this seaweed to produce a large palette of acyclic diterpenes with diverse oxygenation and substitution patterns and promising bioactivities.     

Aqueous Extract of Sea Squirt (Halocynthia roretzi) with Potent Activity against Human Cancer Cells Acts Synergistically with Doxorubicin

Marine ascidian is becoming one of the main sources of an antitumor drug that has shown high bioactivity and extensive application in cancer treatment. Halocynthia roretzi, an edible marine sea squirt, has been demonstrated to have various kinds of biological activities, such as anti-diabetic, anti-hypertension, and enhancing immunity. In this study, we reported that aqueous extracts from the edible parts of H. roretzi presented significantly inhibiting the efficiency on HepG-2 cell viability. The separate mixed compound exhibited strong effects of inhibitory proliferation and induced apoptosis via the generation of ROS along with the concurrent loss of mitochondrial membrane potential on tumor cells. Furthermore, we found that there existed a significantly synergistic effect of the ascidian-extracted compound mixture with the anti-cancer drug doxorubicin. In the presence of the extracts from H. roretzi, the dose of doxorubicin at the cellular level could be reduced by a half dose. The extracts were further divided by semipreparative-HPLC and the active ingredients were identified as a mixture of fatty amide, which was composed of hexadecanamide, stearamide, and erucamide by UHPLC-MS/MS. Our results suggest that the potential toxicity of ascidian H. roretzi in tumor cells, and the compounds extracted from H. roretzi could be potentially utilized on functional nutraceuticals or as an adjunct in combination with chemotherapy.     

Synthesis and Antitumor Activities of Derivatives of the Marine Mangrove Fungal Metabolite Deoxybostrycin

Deoxybostrycin (1) is an anthraquinone compound derived from the marine mangrove fungus Nigrospora sp. No. 1403 and has potential to be a lead for new drugs because of its various biological properties. A series of new derivatives (2–22) of deoxybostrycin were synthesized. The in vitro cytotoxicity of all the new compounds was tested against MDA-MB-435, HepG2 and HCT-116 cancer cell lines. Most of the compounds exhibit strong cytotoxicity with IC₅₀ values ranging from 0.62 to 10 μM. Compounds 19, 21 display comparable cytotoxicity against MDA-MB-435 to epirubicin, the positive control. The primary screening results indicate that the deoxybostrycin derivatives might be a valuable source of new potent anticancer drug candidates.     

Isolation of Scalarane-Type Sesterterpenoids from the Marine Sponge Dysidea sp. and Stereochemical Reassignment of 12-epi-Phyllactone D/E

The chemical investigation of the marine sponge Dysidea sp., which was collected from Bohol province in the Philippines, resulted in the identification of 15 new scalarane-type sesterterpenoids (1–14, 16), together with 15 known compounds. The chemical structures of the new compounds were elucidated based on NMR spectroscopy and HRMS. The structure of 12-epi-phyllactone D/E (15) isolated during this study was originally identified in 2007. However, careful inspection of our experimental ¹³C NMR spectrum revealed considerable discrepancies with the reported data at C-9, C-12, C-14, and C-23, leading to the correction of the reported compound to the C-12 epimer of 15, phyllactone D/E. The biological properties of compounds 1–16 were evaluated using the MDA-MB-231 cancer cell line. Compound 7, which bears a pentenone E-ring, exhibits significant cytotoxicity with a GI₅₀ value of 4.21 μM.     

Bioactive Secondary Metabolites from the Red Sea Marine Verongid Sponge Suberea Species

In a continuation of our efforts to identify bioactive compounds from Red Sea Verongid sponges, the organic extract of the sponge Suberea species afforded seven compounds including two new dibrominated alkaloids, subereamollines C and D (1 and 2), together with the known compounds aerothionin (3), homoaerothionin (4), aeroplysinin-1 (5), aeroplysinin-2 (6) and a revised subereaphenol C (7) as ethyl 2-(2,4-dibromo-3,6-dihydroxyphenyl)acetate. The structures of the isolated compounds were assigned by different spectral data including optical rotations, 1D (¹H and ¹³C) and 2D (COSY, multiplicity-edited HSQC, and HMBC) NMR and high-resolution mass spectroscopy. Aerothionin (3) and subereaphenol C (7) displayed potent cytotoxic activity against HeLa cell line with IC₅₀ values of 29 and 13.3 µM, respectively. In addition, aeroplysinin-2 (6) showed potent antimigratory activity against the human breast cancer cell line MDA-MB-231 with IC₅₀ of 18 µM. Subereamollines C and D are new congeners of the previously reported compounds subereamollines A and B with methyl ester functionalities on the side chain. These findings provide further insight into the biosynthetic capabilities of members of the genus Suberea and the chemical diversity as well as the biological activity of these compounds.     

The Marine-Derived Sipholenol A-4-O-3′,4′-Dichlorobenzoate Inhibits Breast Cancer Growth and Motility in Vitro and in Vivo through the Suppression of Brk and FAK Signaling

Sipholenol A is a natural sipholane triterpenoid isolated from the Red Sea sponge, Callyspongia siphonella. Previous studies showed the antimigratory and antiproliferative activities of the semisynthetic sipholenol A esters against breast cancer cell lines. This study investigated the effects of sipholenol A-4-O-3′,4′-dichlorobenzoate (SPA) on the growth, migration and invasion of diverse human breast cancer cells. Results showed that SPA inhibited the growth of the human breast cancer cells, MDA-MB-231, MCF-7, BT-474 and T-47D, in a dose-dependent manner. Immunofluorescent analysis showed that SPA significantly reduced Ki-67-positive cells in MDA-MB-231 cells. Flow cytometry and Western blot analyses revealed that SPA treatment suppressed MDA-MB-231 cell growth by inducing cell cycle arrest at the G1 phase. In addition, SPA suppressed breast cancer cell migration, invasion and decreased Brk and FAK activation in a dose-dependent manner. Molecular docking study suggested a perfect fitting at the FAK’s FERM domain, inhibiting the main autophosphorylation site, Y397, which was further confirmed by Western blot analysis. Most known small molecule FAK inhibitors target the kinase domain, creating several off-target side effects. The in vivo studies showed that SPA treatment suppressed breast tumor growth and Ki-67, CD31, p-Brk and p-FAK expression in orthotopic breast cancer in nude mice. In conclusion, SPA inhibited the growth, invasion and migration of breast cancer cells possibly via deactivating Brk and FAK signaling, suggesting good potential for therapeutic use to control invasive breast cancer.     

In Vitro and in Vivo Anticancer Activity of Pardaxin against Proliferation and Growth of Oral Squamous Cell Carcinoma

Pardaxin (H-GFFALIPKIISSPLFKTLLSAVGSALSSSGGQE-OH), a 33-amino-acid polypeptide, is an antimicrobial peptide (AMP) isolated from the marine fish species Pardachirus marmoratus. Pardaxin shows antibacterial and antitumor activities. However, pardaxin-induced inhibition of oral cancer and the mechanism of tumor reduction in buccal pouch carcinogenesis after pardaxin painting remain undetermined. Additionally, the toxic effects of pardaxin on normal tissue remain unclear. The present study investigated the anticancer activity of pardaxin in oral squamous cell carcinoma (OSCC) cells in the hamster buccal pouch model with or without 7,12-dimethylbenz[a]anthracene (DMBA) pretreatment. This is the first study to confirm the effects of pardaxin on normal tissue and its nontoxic effects in vivo. Cell viability assays and colony formation tests in OSCC cell lines (SCC-4) demonstrated that pardaxin reduced cell viability in a dose-dependent manner. Immunofluorescence staining of cleaved caspase-3 in SCC-4 cells revealed that expression of activated caspase-3 in SCC-4 cells significantly increased after 24-h treatment with pardaxin. Additionally, a cell cycle analysis indicated that pardaxin treatment resulted in the cell cycle arrest of SCC-4 cells in the G2/M phase, thereby limiting cell proliferation. Furthermore, pardaxin treatment substantially alleviated carcinogenesis in the DMBA-induced hamster buccal pouch model by lowering prostaglandin E₂ levels. These results suggest that pardaxin is a potential marine drug for adjuvant chemotherapy for human OSCC and oral cancer.     

Phallusiasterols A and B: Two New Sulfated Sterols from the Mediterranean Tunicate Phallusia fumigata and Their Effects as Modulators of the PXR Receptor

Purification of the apolar extracts of the marine ascidian Phallusia fumigata, afforded two new sulfated sterols, phallusiasterols A (1) and B (2). The structures of the new compounds have been elucidated using mass spectrometry and NMR experiments. The effects of phallusiasterols A and B as modulators of pregnane-X-receptor (PXR) have been investigated. These studies revealed that phallusiasterol A induces PXR transactivation in HepG2 cells and stimulates the expression of the PXR target genes CYP3A4 and MDR1 in the same cell line. Molecular docking calculations suggested the theoretical binding mode of phallusiasterol A with hPXR and revealed that phallusiasterol A fitted well in the LBD of PXR.     

Antarctic Marine Algae Extracts as a Potential Natural Resource to Protect Epithelial Barrier Integrity

The intestine and skin provide crucial protection against the external environment. Strengthening the epithelial barrier function of these organs is critical for maintaining homeostasis against inflammatory stimuli. Recent studies suggest that polar marine algae are a promising bioactive resource because of their adaptation to extreme environments. To investigate the bioactive properties of polar marine algae on epithelial cells of the intestine and skin, we created extracts of the Antarctic macroalgae Himantothallus grandifolius, Plocamium cartilagineum, Phaeurus antarcticus, and Kallymenia antarctica, analyzed the compound profiles of the extracts using gas chromatography-mass spectrometry, and tested the protective activities of the extracts on human intestinal and keratinocyte cell lines by measuring cell viability and reactive oxygen species scavenging. In addition, we assessed immune responses modulated by the extracts by real-time polymerase chain reaction, and we monitored the barrier-protective activities of the extracts on intestinal and keratinocyte cell lines by measuring transepithelial electrical resistance and fluorescence-labeled dextran flux, respectively. We identified bioactive compounds, including several fatty acids and lipid compounds, in the extracts, and found that the extracts perform antioxidant activities that remove intracellular reactive oxygen species and scavenge specific radicals. Furthermore, the Antarctic marine algae extracts increased cell viability, protected cells against inflammatory stimulation, and increased the barrier integrity of cells damaged by lipopolysaccharide or ultraviolet radiation. These results suggest that Antarctic marine algae have optimized their composition for polar environments, and furthermore, that the bioactive properties of compounds produced by Antarctic marine algae can potentially be used to develop therapeutics to promote the protective barrier function of the intestine and skin.     

Total Synthesis and Biological Activity of Marine Alkaloid Eudistomins Y1–Y7 and Their Analogues

Eudistomin Y class compounds are a series of β-carbolines which was originally isolated from a marine turnicate or ascidian near the South Korea Sea. These compounds contain bromo-substituted groups, which is one of the typical characters of marine natural products. We report herein the chemical synthesis and biological evaluation of seven new β-carboline-based metabolites, Eudistomins Y₁–Y₇, and their hydroxyl-methylated phenyl derivatives. Using bromo-substituted tryptamines and bromo-substituted phenylglyoxals as the key intermediates, Eudistomins Y₁–Y₇ and their derivatives were synthesized via the acid-catalyzed Pictet-Spengler reaction and fully characterized by ¹H- and ¹³C-NMR and mass spectroscopy. Biological studies revealed that all of the compounds showed moderate growth inhibitory activity against breast carcinoma cell line MDA-231 with IC₅₀ of 15–63 μM and the inhibitory activities of hydroxyl-methylated phenyl products were higher than that of the corresponding natural products Eudistomins Y₁–Y₇.     

Polyoxygenated Steroids from the Octocoral Leptogorgia punicea and in Vitro Evaluation of Their Cytotoxic Activity

Five new polyoxygenated marine steroids—punicinols A–E (1–5)—were isolated from the gorgonian Leptogorgia punicea and characterized by spectroscopic methods (IR, MS, ¹H, ¹³C and 2-D NMR). The five compounds induced in vitro cytotoxic effects against lung cancer A549 cells, while punicinols A and B were the most active, with IC₅₀ values of 9.7 μM and 9.6 μM, respectively. The synergistic effects of these compounds with paclitaxel, as well as their effects on cell cycle distribution and their performance in the clonogenic assay, were also evaluated. Both compounds demonstrated significant synergistic effects with paclitaxel.     

Molecular Mechanisms by Which a Fucus vesiculosus Extract Mediates Cell Cycle Inhibition and Cell Death in Pancreatic Cancer Cells

Pancreatic cancer is one of the most aggressive cancer entities, with an extremely poor 5-year survival rate. Therefore, novel therapeutic agents with specific modes of action are urgently needed. Marine organisms represent a promising source to identify new pharmacologically active substances. Secondary metabolites derived from marine algae are of particular interest. The present work describes cellular and molecular mechanisms induced by an HPLC-fractionated, hydrophilic extract derived from the Baltic brown seaweed Fucus vesiculosus (Fv1). Treatment with Fv1 resulted in a strong inhibition of viability in various pancreatic cancer cell lines. This extract inhibited the cell cycle of proliferating cells due to the up-regulation of cell cycle inhibitors, shown on the mRNA (microarray data) and protein level. As a result, cells were dying in a caspase-independent manner. Experiments with non-dividing cells showed that proliferation is a prerequisite for the effectiveness of Fv1. Importantly, Fv1 showed low cytotoxic activity against non-malignant resting T cells and terminally differentiated cells like erythrocytes. Interestingly, accelerated killing effects were observed in combination with inhibitors of autophagy. Our in vitro data suggest that Fv1 may represent a promising new agent that deserves further development towards clinical application.     

Hemimycalins C–E; Cytotoxic and Antimicrobial Alkaloids with Hydantoin and 2-Iminoimidazolidin-4-one Backbones from the Red Sea Marine Sponge Hemimycale sp.

In the course of our continuing efforts to identify bioactive secondary metabolites from Red Sea marine sponges, we have investigated the sponge Hemimycale sp. The cytotoxic fraction of the organic extract of the sponge afforded three new compounds, hemimycalins C–E (1–3). Their structural assignments were obtained via analyses of their one- and two-dimensional NMR spectra and HRESI mass spectrometry. Hemimycalin C was found to differ from the reported hydantoin compounds in the configuration of the olefinic moiety at C-5–C-6, while hemimycalins D and E were found to contain an 2-iminoimidazolidin-4-one moiety instead of the hydantoin moiety in previously reported compounds from the sponge. Hemimycalins C–E showed significant antimicrobial activity against Escherichia coli and Candida albicans and cytotoxic effects against colorectal carcinoma (HCT 116) and the triple-negative breast cancer (MDA-MB-231) cells.