Identification of New EGFR Inhibitors by Structure-Based Virtual Screening and Biological Evaluation.

Epidermal growth factor receptor (EGFR) inhibitors have been used in clinical for the treatment of non-small-cell lung cancer for years. However, the emergence of drug resistance continues to be a major problem. To identify potential inhibitors, molecular docking-based virtual screening was conducted on ChemDiv and Enamine commercial databases using the Glide program. After multi-step VS and visual inspection, a total of 23 compounds with novel and varied structures were selected, and the predicted ADMET properties were within the satisfactory range. Further molecular dynamics simulations revealed that the reprehensive compound ZINC49691377 formed a stable complex with the allosteric pocket of EGFR and exhibited conserved hydrogen bond interactions with Lys 745 and Asp855 of EGFR over the course of simulation. All compounds were further tested in experiments. Among them, the most promising hit ZINC49691377 demonstrated excellent anti-proliferation activity against H1975 and PC-9 cells, while showing no significant anti-proliferation activity against A549 cells. Meanwhile, apoptosis analysis indicated that the compound ZINC49691377 can effectively induce apoptosis of H1975 and PC-9 cells in a dose-dependent manner, while having no significant effect on the apoptosis of A549 cells. The results indicate that ZINC49691377 exhibits good selectivity. Based on virtual screening and bioassays, ZINC4961377 can be considered as an excellent starting point for the development of new EGFR inhibitors.

N-Desmethylmajusculamide B, a lipopeptide isolated from the Okinawan cyanobacterium Okeania hirsuta.

A new lipopeptide, N-desmethylmajusculamide B (1), was isolated from the Okinawan cyanobacterium Okeania hirsuta along with 2 known compounds majusculamide A (2) and majusculamide B (3). The planar structure of (1) was elucidated by a detailed analysis of mass spectrometry and nuclear magnetic resonance spectra. The absolute configurations of the amino acid residues were determined using Marfey's analysis. The configuration of C-16 in the α-methyl-ß-keto-decanoyl moiety was determined unambiguously to be S by conducting a semisynthesis of N-desmethylmajusculamide B from 3. The cytotoxicity against mouse L1210 leukemia cells was evaluated for majusculamides (1-3).

Daboialipase, a phospholipase A2 from Vipera russelli russelli venom posesses anti-platelet, anti-thrombin and anti-cancer properties.

Snake venoms are known to contain toxins capable of interfering with normal physiological processes of victims. Specificity of toxins from snake venoms give scope to identify new molecules with therapeutic action and/or help to understand different cellular mechanisms. Russell's viper venom (RVV) is a mixture of many bioactive molecules with enzymatic and non-enzymatic proteins. The present article describes Daboialipase (DLP), an enzymatic phospholipase A2 with molecular mass of 14.3 kDa isolated from RVV. DLP was obtained after cation exchange chromatography followed by size-exclusion high performance liquid chromatography (SE-HPLC). The isolated DLP presented strong inhibition of adenosine di-phosphate (ADP) and collagen induced platelet aggregation. It also showed anti-thrombin properties by significantly extending thrombin time in human blood samples. Trypan blue and resazurin cell viability assays confirmed time-dependent cytotoxic and cytostatic activities of DLP on MCF7 breast cancer cells, in vitro. DLP caused morphological changes and nuclear damage in MCF7 cells. However, DLP did not cause cytotoxic effects on non-cancer HaCaT cells. Peptide sequences of DLP obtained by O-HRLCMS analysis showed similarity with a previously reported PLA2 (Uniprot ID: PA2B_DABRR/PDB ID: 1VIP_A). An active Asp at 49th position, calcium ion binding site and anticoagulant activity sites were identified in 1 VIP_A. These findings are expected to contribute to designing new anti-platelet, anticoagulant and anti-cancer molecules.

Antihepatoma peptide, scolopentide, derived from the centipede scolopendra subspinipes mutilans.

BACKGROUND: Centipedes have been used to treat tumors for hundreds of years in China. However, current studies focus on antimicrobial and anticoagulation agents rather than tumors. The molecular identities of antihepatoma bioactive components in centipedes have not yet been extensively investigated. It is a challenge to isolate and characterize the effective components of centipedes due to limited peptide purification technologies for animal-derived medicines. AIM: To purify, characterize, and synthesize the bioactive components with the strongest antihepatoma activity from centipedes and determine the antihepatoma mechanism. METHODS: An antihepatoma peptide (scolopentide) was isolated and identified from the centipede scolopendra subspinipes mutilans using a combination of enzymatic hydrolysis, a Sephadex G-25 column, and two steps of high-performance liquid chromatography (HPLC). Additionally, the CCK8 assay was used to select the extracted fraction with the strongest antihepatoma activity. The molecular weight of the extracted scolopentide was characterized by quadrupole time of flight mass spectrometry (QTOF MS), and the sequence was matched by using the Mascot search engine. Based on the sequence and molecular weight, scolopentide was synthesized using solid-phase peptide synthesis methods. The synthetic scolopentide was confirmed by MS and HPLC. The antineoplastic effect of extracted scolopentide was confirmed by CCK8 assay and morphological changes again in vitro. The antihepatoma effect of synthetic scolopentide was assessed by the CCK8 assay and Hoechst staining in vitro and tumor volume and tumor weight in vivo. In the tumor xenograft experiments, qualified model mice (male 5-week-old BALB/c nude mice) were randomly divided into 2 groups (n = 6): The scolopentide group (0.15 mL/d, via intraperitoneal injection of synthetic scolopentide, 500 mg/kg/d) and the vehicle group (0.15 mL/d, via intraperitoneal injection of normal saline). The mice were euthanized by cervical dislocation after 14 d of continuous treatment. Mechanistically, flow cytometry was conducted to evaluate the apoptosis rate of HepG2 cells after treatment with extracted scolopentide in vitro. A Hoechst staining assay was also used to observe apoptosis in HepG2 cells after treatment with synthetic scolopentide in vitro. CCK8 assays and morphological changes were used to compare the cytotoxicity of synthetic scolopentide to liver cancer cells and normal liver cells in vitro. Molecular docking was performed to clarify whether scolopentide tightly bound to death receptor 4 (DR4) and DR5. qRT-PCR was used to measure the mRNA expression of DR4, DR5, fas-associated death domain protein (FADD), Caspase-8, Caspase-3, cytochrome c (Cyto-C), B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), x-chromosome linked inhibitor-of-apoptosis protein and Cellular fas-associated death domain-like interleukin-1ß converting enzyme inhibitory protein in hepatocarcinoma subcutaneous xenograft tumors from mice. Western blot assays were used to measure the protein expression of DR4, DR5, FADD, Caspase-8, Caspase-3, and Cyto-C in the tumor tissues. The reactive oxygen species (ROS) of tumor tissues were tested. RESULTS: In the process of purification, characterization and synthesis of scolopentide, the optimal enzymatic hydrolysis conditions (extract ratio: 5.86%, IC50: 0.310 mg/mL) were as follows: Trypsin at 0.1 g (300 U/g, centipede-trypsin ratio of 20:1), enzymolysis temperature of 46 °C, and enzymolysis time of 4 h, which was superior to freeze-thawing with liquid nitrogen (IC50: 3.07 mg/mL). A peptide with the strongest antihepatoma activity (scolopentide) was further purified through a Sephadex G-25 column (obtained A2) and two steps of HPLC (obtained B5 and C3). The molecular weight of the extracted scolopentide was 1018.997 Da, and the peptide sequence was RAQNHYCK, as characterized by QTOF MS and Mascot. Scolopentide was synthesized in vitro with a qualified molecular weight (1018.8 Da) and purity (98.014%), which was characterized by MS and HPLC. Extracted scolopentide still had an antineoplastic effect in vitro, which inhibited the proliferation of Eca-109 (IC50: 76.27 µg/mL), HepG2 (IC50: 22.06 µg/mL), and A549 (IC50: 35.13 µg/mL) cells, especially HepG2 cells. Synthetic scolopentide inhibited the proliferation of HepG2 cells (treated 6, 12, and 24 h) in a concentration-dependent manner in vitro, and the inhibitory effects were the strongest at 12 h (IC50: 208.11 µg/mL). Synthetic scolopentide also inhibited the tumor volume (Vehicle vs Scolopentide, P = 0.0003) and weight (Vehicle vs Scolopentide, P = 0.0022) in the tumor xenograft experiment. Mechanistically, flow cytometry suggested that the apoptosis ratios of HepG2 cells after treatment with extracted scolopentide were 5.01% (0 µg/mL), 12.13% (10 µg/mL), 16.52% (20 µg/mL), and 23.20% (40 µg/mL). Hoechst staining revealed apoptosis in HepG2 cells after treatment with synthetic scolopentide in vitro. The CCK8 assay and morphological changes indicated that synthetic scolopentide was cytotoxic and was significantly stronger in HepG2 cells than in L02 cells. Molecular docking suggested that scolopentide tightly bound to DR4 and DR5, and the binding free energies were-10.4 kcal/mol and-7.1 kcal/mol, respectively. In subcutaneous xenograft tumors from mice, quantitative real-time polymerase chain reaction and western blotting suggested that scolopentide activated DR4 and DR5 and induced apoptosis in SMMC-7721 Liver cancer cells by promoting the expression of FADD, caspase-8 and caspase-3 through a mitochondria-independent pathway. CONCLUSION: Scolopentide, an antihepatoma peptide purified from centipedes, may inspire new antihepatoma agents. Scolopentide activates DR4 and DR5 and induces apoptosis in liver cancer cells through a mitochondria-independent pathway.

Anticancer effects of the HDAC inhibitor, 3ß,6ß­dihydroxyurs­12­en­27­oic acid, in MCF­7 breast cancer cells via the inhibition of Akt/mTOR pathways.

Astilbe chinensis (A. chinensis) is a perennial herb that is used to treat chronic bronchitis and pain. The anticancer activity of 3ß,6ß­dihydroxyurs­12­en­27­oic acid (ACT­3), a major component isolated from A. chinensis, has not yet been investigated in detail. The purpose of the present study was to investigate the histone deacetylase (HDAC) inhibitory and anticancer activities of ACT­3 compared with suberoylanilide hydroxamic acid (SAHA) in MCF­7 human breast cancer cells. The purity of ACT­3 was determined using high­performance liquid chromatography. In the present study, the effects of ACT­3 on anticancer effects of MCF­7 cells were determined by measuring the level of apoptotic cell death and cell cycle regulator using flow cytometry analysis and western blot analysis, respectively. The effects of ACT­3 on HDAC enzyme activity were measured using assay kits. ACT­3 and SAHA increased the levels of acetylated histone H3 and reduced the levels of HDAC1 and HDAC3 in MCF­7 cells. ACT­3 significantly decreased the cell viability in a concentration­dependent manner and induced different morphological changes at high concentrations. ACT­3 and SAHA significantly inhibited the colony formation in MCF­7 cells. ACT­3 inhibited total HDAC activity in a dose­dependent manner. ACT­3 significantly reduced the expression levels of cyclin D1 and cyclin­dependent kinase 4, and upregulated the expression levels of p21WAF1 and p53. A significant increase in the G1 phase cell population was observed in MCF­7 cells and ACT­3 induced apoptosis by reducing the ratio of B­cell lymphoma­2 (Bcl­2)/Bcl­2­associated X (Bax) and releasing cleaved caspase 9. Additionally, ACT­3 significantly increased autophagic cell death by inhibiting the serine­threonine kinase/mammalian target of the rapamycin pathway. Autophagy induction was confirmed via acridine orange staining. ACT­3 significantly increased the pERK1/2 and p21 in MCF­7 cells. Thus, the activated ERK pathway played an important role in cell cycle arrest and apoptosis via ERK­dependent induction of p21 in MCF­7 cells. These data indicated that ACT­3 can be used as a promising anticancer agent to overcome the limitations and reduce the side effects of conventional anticancer drugs.

The Pharmacological Potential of Novel Melittin Variants from the Honeybee and Solitary Bees against Inflammation and Cancer.

The venom of honeybees is composed of numerous peptides and proteins and has been used for decades as an anti-inflammatory and anti-cancer agent in traditional medicine. However, the bioactivity of specific biomolecular components has been evaluated for the predominant constituent, melittin. So far, only a few melittin-like peptides from solitary bee species have been investigated, and the molecular mechanisms of bee venoms as therapeutic agents remain largely unknown. Here, the preclinical pharmacological activities of known and proteo-transcriptomically discovered new melittin variants from the honeybee and more ancestral variants from phylogenetically older solitary bees were explored in the context of cancer and inflammation. We studied the effects of melittin peptides on cytotoxicity, second messenger release, and inflammatory markers using primary human cells, non-cancer, and cancerous cell lines. Melittin and some of its variants showed cytotoxic effects, induced Ca2+ signaling and inhibited cAMP production, and prevented LPS-induced NO synthesis but did not affect the IP3 signaling and pro-inflammatory activation of endothelial cells. Compared to the originally-described melittin, some phylogenetically more ancestral variants from solitary bees offer potential therapeutic modalities in modulating the in vitro inflammatory processes, and hindering cancer cell viability/proliferation, including aggressive breast cancers, and are worth further investigation.

A Dimerosesquiterpene and Sesquiterpene Lactones from Artemisia argyi Inhibiting Oncogenic PI3K/AKT Signaling in Melanoma Cells.

A library of more than 2500 plant extracts was screened for activity on oncogenic signaling in melanoma cells. The ethyl acetate extract from the aerial parts of Artemisia argyi displayed pronounced inhibition of the PI3K/AKT pathway. Active compounds were tracked with the aid of HPLC-based activity profiling, and altogether 21 active compounds were isolated, including one novel dimerosequiterpenoid (1), one new disesquiterpenoid (2), three new guaianolides (3-5), 12 known sesquiterpenoids (6-17), and four known flavonoids (19-22). A new eudesmanolide derivative (13b) was isolated as an artifact formed by methanolysis. Compound 1 is the first adduct comprising a sesquiterpene lactone and a methyl jasmonate moiety. The absolute configurations of compounds 1 and 3-18 were established by comparison of their experimental and calculated ECD spectra. The absolute configuration for 2 was determined by X-ray diffraction analysis. Guaianolide 8 was the most potent sesquiterpene lactone, inhibiting the PI3K/AKT pathway with an IC50 value of 8.9 ± 0.9 µM.

Cladoxanthones A and B, Xanthone-Derived Metabolites with a Spiro[cyclopentane-1,2'-[3,9a]ethanoxanthene]-2,4',9',11'-tetraone Skeleton from a Cladosporium sp.

Cladoxanthones A (1) and B (2), two xanthone-derived metabolites featuring a new spiro[cyclopentane-1,2'-[3,9a]ethanoxanthene]-2,4',9',11'(4a'H)-tetraone skeleton, were isolated from cultures of the ascomycete fungus Cladosporium sp., together with the known mangrovamide J (3). Their structures were elucidated primarily by NMR experiments. The absolute configurations of 1 and 2 were assigned by X-ray crystallography using Cu Kα radiation. Compound 1 could be generated from the hypothetical precursors related to α-methylene ketone and dihydro-xanthone via a Diels-Alder reaction, while 2 could be an oxidative coupling product resulting from 1 and 3. Compounds 1 and 2 showed weakly cytotoxic effects.

Cytotoxic Cyclodepsipeptides and Cyclopentane Derivatives from a Plant-Associated Fungus Fusarium sp.

In this work, four new cyclodepsipeptides, fusarihexins C-E (1-3) and enniatin Q (4), four new cyclopentane derivatives, fusarilins A-D (5-8), together with eight known compounds (9-16), were isolated from cultures of the endophytic fungus Fusarium sp. The structures of the isolated compounds were elucidated by analysis of HRMS and NMR spectroscopic data. The absolute configurations were determined using Marfey's method, a modified Mosher's method, single-crystal X-ray diffraction analysis, and ECD analysis. The antitumor activities of the isolated compounds in vitro were evaluated. Cyclodepsipeptides displayed cytotoxicities against the Huh-7, MRMT-1, and HepG-2 cell lines. Compounds 4, 9, 10, and 12 with IC50 values of 1.0-9.1 µM exhibited the most potent cytotoxicities against the three cell lines as compared to the positive control-5-fluorouracil. Compounds 1-3 and 11 exhibited moderate cytotoxic activities (IC50 values of 10.7-20.1 µM).

An immunogenic cell injury module for the single-cell multiplexed activity metabolomics platform to identify promising anti-cancer natural products.

Natural products constitute and significantly impact many current anti-cancer medical interventions. A subset of natural products induces injury processes in malignant cells that recruit and activate host immune cells to produce an adaptive anti-cancer immune response, a process known as immunogenic cell death. However, a challenge in the field is to delineate forms of cell death and injury that best promote durable antitumor immunity. Addressing this with a single-cell chemical biology natural product discovery platform, like multiplex activity metabolomics, would be especially valuable in human leukemia, where cancer cells are heterogeneous and may react differently to the same compounds. Herein, a new ten-color, fluorescent cell barcoding-compatible module measuring six immunogenic cell injury signaling readouts are as follows: DNA damage response (γH2AX), apoptosis (cCAS3), necroptosis (p-MLKL), mitosis (p-Histone H3), autophagy (LC3), and the unfolded protein response (p-EIF2α). A proof-of-concept screen was performed to validate functional changes in single cells induced by secondary metabolites with known mechanisms within bacterial extracts. This assay was then applied in multiplexed activity metabolomics to reveal an unexpected mammalian cell injury profile induced by the natural product narbomycin. Finally, the functional consequences of injury pathways on immunogenicity were compared with three canonical assays for immunogenic hallmarks, ATP, HMGB1, and calreticulin, to correlate secondary metabolite-induced cell injury profiles with canonical markers of immunogenic cell death. In total, this work demonstrated a new phenotypic screen for discovery of natural products that modulate injury response pathways that can contribute to cancer immunogenicity.

Discovery of potent BET bromodomain 1 stereoselective inhibitors using DNA-encoded chemical library selections.

BRDT, BRD2, BRD3, and BRD4 comprise the bromodomain and extraterminal (BET) subfamily which contain two similar tandem bromodomains (BD1 and BD2). Selective BD1 inhibition phenocopies effects of tandem BET BD inhibition both in cancer models and, as we and others have reported of BRDT, in the testes. To find novel BET BD1 binders, we screened >4.5 billion molecules from our DNA-encoded chemical libraries with BRDT-BD1 or BRDT-BD2 proteins in parallel. A compound series enriched only by BRDT-BD1 was resynthesized off-DNA, uncovering a potent chiral compound, CDD-724, with >2,000-fold selectivity for inhibiting BRDT-BD1 over BRDT-BD2. CDD-724 stereoisomers exhibited remarkable differences in inhibiting BRDT-BD1, with the R-enantiomer (CDD-787) being 50-fold more potent than the S-enantiomer (CDD-786). From structure­activity relationship studies, we produced CDD-956, which maintained picomolar BET BD1 binding potency and high selectivity over BET BD2 proteins and had improved stability in human liver microsomes over CDD-787. BROMOscan profiling confirmed the excellent pan-BET BD1 affinity and selectivity of CDD-787 and CDD-956 on BD1 versus BD2 and all other BD-containing proteins. A cocrystal structure of BRDT-BD1 bound with CDD-956 was determined at 1.82 Å and revealed BRDT-BD1­specific contacts with the αZ and αC helices that explain the high affinity and selectivity for BET BD1 versus BD2. CDD-787 and CDD-956 maintain cellular BD1-selectivity in NanoBRET assays and show potent antileukemic activity in acute myeloid leukemia cell lines. These BET BD1-specific and highly potent compounds are structurally unique and provide insight into the importance of chirality to achieve BET specificity.

In Vitro Anticancer and Cancer-Preventive Activity of New Triterpene Glycosides from the Far Eastern Starfish Solaster pacificus.

Sea stars or starfish (class Asteroidea) and holothurians or sea cucumbers (class Holothuroidea), belonging to the phylum Echinodermata (echinoderms), are characterized by different sets of glycosidic metabolites: the steroid type in starfish and the triterpene type in holothurians. However, herein we report the isolation of eight new triterpene glycosides, pacificusosides D−K (1−3, 5−9) along with the known cucumarioside D (4), from the alcoholic extract of the Far Eastern starfish Solaster pacificus. The isolated new compounds are closely related to the metabolites of sea cucumbers, and their structures of 1−3 and 5−9 were determined by extensive NMR and ESIMS techniques. Compounds 2, 5, and 8 have a new type of tetrasaccharide chain with a terminal non-methylated monosaccharide unit. Compounds 3, 6, and 9 contain another new type of tetrasaccharide chain, having 6-O-SO3-Glc as one of the sugar units. The cytotoxic activity of 1−9 against non-cancerous mouse epidermal cells JB6 Cl41 and human melanoma cell lines SK-MEL-2, SK-MEL-28, and RPMI-7951 was determined by MTS assay. Compounds 1, 3, 4, 6, and 9 showed potent cytotoxicity against these cell lines, but the cancer selectivity (SI > 9) was observed only against the SK-MEL-2 cell line. Compounds 1, 3, 4, 6, and 9 at the non-toxic concentration of 0.1 µM significantly inhibited neoplastic cell transformation of JB6 Cl41 cells induced by chemical carcinogens (EGF, TPA) or ionizing radiation (X-rays and UVB). Moreover, compounds 1 and 4 at the non-toxic concentration of 0.1 µM possessed the highest inhibiting activity on colony formation among the investigated compounds and decreased the colonies number of SK-MEL-2 cells by 64% and 70%, respectively. Thus, triterpene glycosides 1 and 4 can be considered as prospective cancer-preventive and anticancer-compound leaders.

Disulfated Ophiuroid Type Steroids from the Far Eastern Starfish Pteraster marsippus and Their Cytotoxic Activity on the Models of 2D and 3D Cultures.

New steroidal 3ß,21-disulfates (2-4), steroidal 3ß,22-disulfate (5), and the previously known related steroidal 3ß,21-disulfate (1) were isolated from the ethanolic extract of the Far Eastern starfish Pteraster marsippus, collected off Urup Island in the Sea of Okhotsk. The structures of these compounds were determined by intensive NMR and HRESIMS techniques as well as by chemical transformations. Steroids 2 and 3 have an oxo-group in the tetracyclic nucleus at position C-7 and differ from each other by the presence of the 5(6)-double bond. The Δ24-22-sulfoxycholestane side chain of the steroid 5 has not been found previously in the starfish or ophiuroid steroids. The cytotoxic activities of 1, 4, 5, and the mixture of 2 and 3 were determined on the models of 2D and 3D cultures of human epithelial kidney cells (HEK293), melanoma cells (SK-MEL-28), small intestine carcinoma cells (HuTu80), and breast carcinoma cells (ZR-75-1). The mixture of 2 and 3 revealed a significant inhibitory effect on the cell viability of human breast carcinoma ZR-75-1 cells, but other tested compounds were less effective.

Elucidation of the Metabolite Profile of Yucca gigantea and Assessment of Its Cytotoxic, Antimicrobial, and Anti-Inflammatory Activities.

The acute inflammation process is explained by numerous hypotheses, including oxidative stress, enzyme stimulation, and the generation of pro-inflammatory cytokines. The anti-inflammatory activity of Yucca gigantea methanol extract (YGME) against carrageenan-induced acute inflammation and possible underlying mechanisms was investigated. The phytochemical profile, cytotoxic, and antimicrobial activities were also explored. LC-MS/MS was utilized to investigate the chemical composition of YGME, and 29 compounds were tentatively identified. In addition, the isolation of luteolin-7-O-ß-d-glucoside, apigenin-7-O-ß-d-glucoside, and kaempferol-3-O-α-l-rhamnoside was performed for the first time from the studied plant. Inflammation was induced by subcutaneous injection of 100 µL of 1% carrageenan sodium. Rats were treated orally with YGME 100, 200 mg/kg, celecoxib (50 mg/kg), and saline, respectively, one hour before carrageenan injection. The average volume of paws edema and weight were measured at several time intervals. Levels of NO, GSH, TNF-α, PGE-2, serum IL-1ß, IL-6 were measured. In additionally, COX-2 immunostaining and histopathological examination of paw tissue were performed. YGME displayed a potent anti-inflammatory influence by reducing paws edema, PGE-2, TNF-α, NO production, serum IL-6, IL-1ß, and COX-2 immunostaining. Furthermore, it replenished the diminished paw GSH contents and improved the histopathological findings. The best cytotoxic effect of YGME was against human melanoma cell line (A365) and osteosarcoma cell line (MG-63). Moreover, the antimicrobial potential of the extract was evaluated against bacterial and fungal isolates. It showed potent activity against Gram-negative, Gram-positive, and fungal Candida albicans isolates. The promoting multiple effects of YGME could be beneficial in the treatment of different ailments based on its anti-inflammatory, antimicrobial, and cytotoxic effects.

Bursatella leachii Purple Ink Secretion Concentrate Exerts Cytotoxic Properties against Human Hepatocarcinoma Cell Line (HepG2): In Vitro and In Silico Studies.

Liver cancer is a leading cause of cancer death globally. Marine mollusc-derived drugs have gained attention as potential natural-based anti-cancer agents to overcome the side effects caused by conventional chemotherapeutic drugs during cancer therapy. Using liquid chromatography-mass spectrometry, the main biomolecules in the purple ink secretion released by the sea hare, named Bursatella leachii (B. leachii), were identified as hectochlorin, malyngamide X, malyngolide S, bursatellin and lyngbyatoxin A. The cytotoxic effects of B. leachii ink concentrate against human hepatocarcinoma (HepG2) cells were determined to be dose- and time-dependent, and further exploration of the underlying mechanisms causing the programmed cell death (apoptosis) were performed. The expression of cleaved-caspase-8 and cleaved-caspase-3, key cysteine-aspartic proteases involved in the initiation and completion of the apoptosis process, appeared after HepG2 cell exposure to the B. leachii ink concentrate. The gene expression levels of pro-apoptotic BAX, TP53 and Cyclin D1 were increased after treatment with the B. leachii ink concentrate. Applying in silico approaches, the high scores predicted that bioactivities for the five compounds were protease and kinase inhibitors. The ADME and cytochrome profiles for the compounds were also predicted. Altogether, the B. leachii ink concentrate has high pro-apoptotic potentials, suggesting it as a promising safe natural product-based drug for the treatment of liver cancer.

New Trichothecenes Isolated from the Marine Algicolous Fungus Trichoderma brevicompactum.

Eight trichothecenes, including four new compounds 1-4 and four known entities 5-8, together with one known cyclonerane (9) were isolated from the solid-state fermentation of Trichoderma brevicompactum NTU439 isolated from the marine alga Mastophora rosea. The structures of 1-9 were determined by 1D/2D NMR (nuclear magnetic resonance), MS (mass spectrometry), and IR (infrared spectroscopy) spectroscopic data. All of the compounds were evaluated for cytotoxic activity against HCT-116, PC-3, and SK-Hep-1 cancer cells by the SRB assay, and compound 8 showed promising cytotoxic activity against all three cancer cell lines with the IC50 values of 3.3 ± 0.3, 5.3 ± 0.3, and 1.8 ± 0.8 µM, respectively. Compounds 1-2, 4-6, and 7-8 potently inhibited LPS-induced NO production, and compounds 5 and 8 showed markedly inhibited gelatinolysis of MMP-9 in S1 protein-stimulated THP-1 monocytes.

Ligiamycins A and B, Decalin-Amino-Maleimides from the Co-Culture of Streptomyces sp. and Achromobacter sp. Isolated from the Marine Wharf Roach, Ligia exotica.

Streptomyces sp. GET02.ST and Achromobacter sp. GET02.AC were isolated together from the gut of the wharf roach, Ligia exotica, inhabiting the intertidal zone of the west coast of Korea. The co-cultivation of these two strains significantly induced the production of two new metabolites, ligiamycins A (1) and B (2), which were barely detected in the single culture of Streptomyces sp. GET02.ST. The planar structures of ligiamycins A (1) and B (2) were elucidated as new decalins coupled with amino-maleimides by the analysis of various spectroscopic data, including nuclear magnetic resonance (NMR), ultraviolet (UV), and mass (MS) data. The assignment of two nitrogen atoms in amino-maleimide in 1 was accomplished based on 1H-15N heteroatom single quantum coherence spectroscopy (HSQC) NMR experiments. The relative configurations of the ligiamycins were determined using rotating frame Overhauser effect spectroscopy (ROESY) NMR data, and their absolute configurations were deduced by comparing their experimental and calculated optical rotations. Ligiamycin A (1) displayed antibacterial effects against Staphylococcus aureus and Salmonella enterica, while ligiamycin B (2) exhibited mild cell cytotoxicity against human colorectal cancer cells.

Biological Activity and Stability of Aeruginosamides from Cyanobacteria.

Aeruginosamides (AEGs) are classified as cyanobactins, ribosomally synthesized peptides with post-translational modifications. They have been identified in cyanobacteria of genera Microcystis, Oscillatoria, and Limnoraphis. In this work, the new data on the in vitro activities of three AEG variants, AEG A, AEG625 and AEG657, and their interactions with metabolic enzymes are reported. Two aeruginosamides, AEG625 and AEG657, decreased the viability of human breast cancer cell line T47D, but neither of the peptides was active against human liver cancer cell line Huh7. AEGs also did not change the expression of MIR92b-3p, but for AEG625, the induction of oxidative stress was observed. In the presence of a liver S9 fraction containing microsomal and cytosolic enzymes, AEG625 and AEG657 showed high stability. In the same assays, quick removal of AEG A was recorded. The peptides had mild activity against three cytochrome P450 enzymes, CYP2C9, CYP2D6 and CYP3A4, but only at the highest concentration used in the study (60 µM). The properties of AEGs, i.e., cytotoxic activity and in vitro interactions with important metabolic enzymes, form a good basis for further studies on their pharmacological potential.

(-)-Agelasidine A Induces Endoplasmic Reticulum Stress-Dependent Apoptosis in Human Hepatocellular Carcinoma.

Liver cancers, such as hepatocellular carcinoma (HCC), are a highly prevalent cause of cancer-related deaths. Current treatments to combat liver cancer are limited. (-)-Agelasidine A, a compound isolated from the methanol extract of Agelasnakamurai, a sesquiterpene guanidine derived from sea sponge, has antibacterial activity. We demonstrated its anticancer capabilities by researching the associated mechanism of (-)-agelasidine A in human liver cancer cells. We found that (-)-agelasidine A significantly reduced viability in Hep3B and HepG2 cells, and we determined that apoptosis was involved in the (-)-agelasidine A-induced Hep3B cell deaths. (-)-Agelasidine A activated caspases 9, 8, and 3, as well as PARP. This effect was reversed by caspase inhibitors, suggesting caspase-mediated apoptosis in the (-)-agelasidine A-treated Hep3B cells. Moreover, the reduced mitochondrial membrane potential (MMP) and the release of cytochrome c indicated that the (-)-agelasidine A-mediated mitochondrial apoptosis was mechanistic. (-)-Agelasidine A also increased apoptosis-associated proteins (DR4, DR5, FAS), which are related to extrinsic pathways. These events were accompanied by an increase in Bim and Bax, proteins that promote apoptosis, and a decrease in the antiapoptotic protein, Bcl-2. Furthermore, our results presented that (-)-agelasidine A treatment bridged the intrinsic and extrinsic apoptotic pathways. Western blot analysis of Hep3B cells treated with (-)-agelasidine A showed that endoplasmic reticulum (ER) stress-related proteins (GRP78, phosphorylated PERK, phosphorylated eIF2α, ATF4, truncated ATF6, and CHOP) were upregulated. Moreover, 4-PBA, an ER stress inhibitor, could also abrogate (-)-agelasidine A-induced cell viability reduction, annexin V+ apoptosis, death receptor (DR4, DR5, FAS) expression, mitochondrial dysfunction, and cytochrome c release. In conclusion, by activating ER stress, (-)-agelasidine A induced the extrinsic and intrinsic apoptotic pathways of human HCC.

A Novel Peptide Derived from Arca inflata Induces Apoptosis in Colorectal Cancer Cells through Mitochondria and the p38 MAPK Pathway.

Colorectal carcinoma (CRC) is one of the major causes of cancer-related incidence and deaths. Here, we identified a novel antitumor peptide, P6, with a molecular weight of 2794.8 Da from a marine Chinese medicine, Arca inflata Reeve. The full amino acid sequence and secondary structure of P6 were determined by tandem mass de novo sequencing and circular dichroism spectroscopy, respectively. P6 markedly inhibited cell proliferation and colony formation, and induced apoptosis in CRC cells. Mechanistically, transcriptomics analysis and a serial functional evaluation showed that P6 induced colon cancer cell apoptosis through the activation of the p38-MAPK signaling pathway. Moreover, it was demonstrated that P6 exhibited antitumor effects in a tumor xenograft model, and induced cell cycle arrest in CRC cells in a concentration-dependent mode. These findings provide the first line of indication that P6 could be a potential therapeutic agent for CRC treatment.