Evaluation of In Vitro Cytotoxic Potential of Avarol towards Human Cancer Cell Lines and In Vivo Antitumor Activity in Solid Tumor Models.

The goal of this study was to determine the activity in vitro and in vivo of avarol, a sesquiterpene hydroquinone originating from the Dysidea avara sponge from the south Adriatic Sea, against different cancer cell lines and two types of mouse carcinoma. To investigate the in vitro cytotoxicity, a human cervix adenocarcinoma cell line (HeLa), human colon adenocarcinoma (LS174), human non-small-cell lung carcinoma (A549), and a normal human fetal lung fibroblast cell line (MRC-5) were used. The in vivo antitumor activity was investigated against two transplantable mouse tumors, the Ehrlich carcinoma (EC) and cervical cancer (CC-5). The effect of avarol on cancer cell survival, which was determined by the microculture tetrazolium test, confirmed a significant in vitro potency of avarol against the investigated cell lines, without selectivity towards MRC-5. The highest cytotoxicity was exhibited against HeLa cancer cells (10.22 ± 0.28 µg/mL). Moreover, potent antitumor activity against two tumor models was determined, as the intraperitoneal administration of avarol at a dose of 50 mg/kg resulted in a significant inhibition of tumor growth in mice. After three administrations of avarol, a 29% inhibition of the EC growth was achieved, while in the case of CC-5, a 36% inhibition of the tumor growth was achieved after the second administration of avarol. Therefore, the results indicate that this marine sesquiterpenoid hydroquinone could be a promising bioactive compound in the development of new anticancer medicine.

New NF-κB Inhibitory Steroids from the Marine Sponge Dysidea avara Collected from the South China Sea.

Chemical investigation of the marine sponge Dysidea avara, collected from the South China Sea, yielded 13 steroids, including nine new (1-9) and four known (10-13) ones. The new structures were elucidated as (3S,14R)-3,14-dihydroxycholesta-5,8-dien-7-one (1), (22E,24R)-7α-ethoxy-5α,6α-epoxyergosta-8(14),22-dien-3ß-ol (2), 3ß-hydroxy-7α-ethoxy-5α,6α-epoxy-8(14)-cholestene (3), 3ß,5α-dihydroxy-6α-ethoxychofesta-7,9(11)-diene (4), 3ß,5α-dihydroxy-6ß-ethoxycholest-7-ene (5), (22E,24R)-24-ethoxy-3ß,5α-dihydroxy-6ß-ethoxyergosta-7,22-diene (6), (22E)-3ß,5α-dihydroxy-6ß-ethoxycholesta-7,22-diene (7), 24-ethoxy-3ß,5α-dihydroxy-6ß-ethoxycholest-7-ene (8 and 9), by extensive spectroscopic analyses, such as HR-ESI-MS, 1D and 2D NMR data. The absolute configuration of 1 was assigned by comparison the experimental ECD spectra with the calculated ones. Among the 13 metabolites, compounds 1, 4, 11, 12, and 13 showed NF-κB inhibitory activities in human HER-293 cells with IC50 values of 6.4, 18.7, 8.1, 9.6, and 7.5 µM, respectively. Preliminary structure-activity relationship analysis unveiled that the conjugated ketones or unsaturated double bonds might be the functional groups for the five active steroids.

Investigating the Antiparasitic Potential of the Marine Sesquiterpene Avarone, Its Reduced form Avarol, and the Novel Semisynthetic Thiazinoquinone Analogue Thiazoavarone.

The chemical analysis of the sponge Dysidea avara afforded the known sesquiterpene quinone avarone, along with its reduced form avarol. To further explore the role of the thiazinoquinone scaffold as an antiplasmodial, antileishmanial and antischistosomal agent, we converted the quinone avarone into the thiazinoquinone derivative thiazoavarone. The semisynthetic compound, as well as the natural metabolites avarone and avarol, were pharmacologically investigated in order to assess their antiparasitic properties against sexual and asexual stages of Plasmodium falciparum, larval and adult developmental stages of Schistosoma mansoni (eggs included), and also against promastigotes and amastigotes of Leishmania infantum and Leishmania tropica. Furthermore, in depth computational studies including density functional theory (DFT) calculations were performed. A toxic semiquinone radical species which can be produced starting both from quinone- and hydroquinone-based compounds could mediate the anti-parasitic effects of the tested compounds.

Liposomal integration method for assessing antioxidative activity of water insoluble compounds towards biologically relevant free radicals: example of avarol.

The liposomal integration method, in conjunction with electron paramagnetic resonance (EPR) spectroscopy, has been presented for the investigation of antioxidant activity of selected water-insoluble compound towards biologically relevant free radicals. This method was applied to avarol, a sesquiterpenoid hydroquinone isolated from the marine sponge Dysidea avara. The antioxidant activity of water-insoluble avarol towards •OH, O2•- and NO• radicals was attained by its incorporation into the DPPC liposomes bilayer, and towards ascorbyl radicals in the organic solvent. Avarol's activity towards •OH, O2•-, NO• and ascorbyl radicals was 86.2%, 50.9%, 23.6% and 61.8%, respectively, showing its significant radical scavenging potential.

Further in vitro biological activity evaluation of amino-, thio- and ester-derivatives of avarol.

The acetylcholinesterase inhibitory and/or antitumour activities of amino-, thio- and ester-derivatives of avarol selected were evaluated for the first time at in vitro conditions. Avarol-3',4'-dithioglycol (1) and avarol-4'-(3)mercaptopropionic acid (3) were shown to be the best inhibitors of the enzyme tested (0.50 µg and IC50 0.05 mM and 0.50 µg and IC50 0.12 mM, respectively), while 4'-tryptamine-avarone (9) and avarol-3'-(3)mercaptopropionic acid (2) exhibited the highest cytotoxicity against the human breast T-47D cancer cell line (IC50 0.66 µg/mL and 1.25 µg/mL, respectively). According to experimental data obtained, the sesquiterpenoid hydroquinone structure of bioactive avarol derivatives may inspire development of new pharmacologically useful substances to be used in the treatment of Alzheimer's disease and/or human breast tumour.

Further in vitro biological activity evaluation of amino-, thio- and ester-derivatives of avarol.

The acetylcholinesterase inhibitory and/or antitumour activities of amino-, thio- and ester-derivatives of avarol selected were evaluated for the first time at in vitro conditions. Avarol-3',4'-dithioglycol (1) and avarol-4'-(3)mercaptopropionic acid (3) were shown to be the best inhibitors of the enzyme tested (0.50 µg and IC50 0.05 mM and 0.50 µg and IC50 0.12 mM, respectively), while 4'-tryptamine-avarone (9) and avarol-3'-(3)mercaptopropionic acid (2) exhibited the highest cytotoxicity against the human breast T-47D cancer cell line (IC50 0.66 µg/mL and 1.25 µg/mL, respectively). According to experimental data obtained, the sesquiterpenoid hydroquinone structure of bioactive avarol derivatives may inspire development of new pharmacologically useful substances to be used in the treatment of Alzheimer's disease and/or human breast tumour.

Evidence of Insulin-Sensitizing and Mimetic Activity of the Sesquiterpene Quinone Avarone, a Protein Tyrosine Phosphatase 1B and Aldose Reductase Dual Targeting Agent from the Marine Sponge Dysidea avara.

Type 2 diabetes mellitus (T2DM) is a complex disease characterized by impaired glucose homeostasis and serious long-term complications. First-line therapeutic options for T2DM treatment are monodrug therapies, often replaced by multidrug therapies to ensure that non-responding patients maintain target glycemia levels. The use of multitarget drugs instead of mono- or multidrug therapies has been emerging as a main strategy to treat multifactorial diseases, including T2DM. Therefore, modern drug discovery in its early stages aims to identify potential modulators for multiple targets; for this purpose, exploration of the chemical space of natural products represents a powerful tool. Our study demonstrates that avarone, a sesquiterpene quinone obtained from the sponge Dysidea avara, is capable of inhibiting in vitro PTP1B, the main negative regulator of the insulin receptor, while it improves insulin sensitivity, and mitochondria activity in C2C12 cells. We observe that when avarone is administered alone, it acts as an insulin-mimetic agent. In addition, we show that avarone acts as a tight binding inhibitor of aldose reductase (AKR1B1), the enzyme involved in the development of diabetic complications. Overall, avarone could be proposed as a novel natural hit to be developed as a multitarget drug for diabetes and its pathological complications.

Dysideanones F-G and dysiherbols D-E, unusual sesquiterpene quinones with rearranged skeletons from the marine sponge Dysidea avara.

Four new sesquiterpene quinone meroterpenoids, dysideanones F-G (1-2) and dysiherbols D-E (3-4), were isolated from the marine sponge Dysidea avara collected from the South China Sea. The new structures were elucidated by extensive analysis of spectroscopic data including HR-MS and 1D and 2D NMR spectra, and their absolute configurations were assigned by single-crystal X-ray diffraction and ECD calculations. Anti-inflammatory evaluation showed that dysiherbols D-E (3-4) exhibited moderate inhibitory activity on TNF-α-induced NF-κB activation in human HEK-293T cells with IC50 values of 10.2 and 8.6 µmol·L-1, respectively.

Isolation, identification, and HPTLC quantification of dehydrodeoxycholic acid from Persian Gulf sponges.

This study aims to investigate the chemical constituents of sponges Dysidea avara (D. avara) and Axinella sinoxea (A. sinoxea), grown up in the Persian Gulf, as well as dehydrodeoxycholic acid (DHCA) content in methanolic extracts of the selected sponges. The chromatography-mass spectrometry (GC-MS) fingerprint of bioactive compounds from methanolic extracts of the selected marine sponge samples was investigated. Based on molecular docking results, among chemical compounds found in marine sponges, DHCA has anti-inflammatory and antipsoriatic properties. They also indicated that DHCA generated stable complexes with 1w81, 3bqm, and 3k8o receptors (psoriasis-related targets) with a binding energy (BE) of -9.26, -10.62, and -7.59 kcal mol-1, respectively. DHCA is isolated from the methanolic extracts of marine sponge samples on chromatographic plates was quantified after derivatization with anisaldehyde reagent by the validated HPTLC method. In-situ HPTLC-DPPH was also calculated to evaluate the free radical-scavenging activity (FRSA) of DHCA. In-silico ADME (Absorption, Distribution, Metabolism, Excretion) predictions revealed that the compound had minimum toxicity and acceptable human intestinal absorption (HIA), as well as low skin permeability. These can potentially be employed as lead compounds to develop a novel antipsoriatic drug.

An "olivomycin A" derivative from a sponge-associated Streptomyces sp. strain SP 85.

We isolated an actinobacterium, Streptomyces sp. strain SP 85 from the marine sponge Dysidea avara. Polyphasic identification of the microorganism showed that the strain SP 85 had high 16S rRNA gene similarity (99%) with Streptomyces olivaceus strain NBRC 12805, while some physiological and biochemical differences were observed. A cytotoxic compound, SP 85 was isolated from the active culture extract of the strain SP 85 by bioassay-guided purification over silica gel column chromatography, preparative TLC, and HPLC. The structure elucidation based on the spectroscopic analysis, including UV, ESI-MS, and 13C NMR data revealed that SP 85 compound is an analog of anti-tumor drug, "olivomycin A". The SP 85 compound showed high cytotoxic activity against three human cancer cell lines, including SW480, HepG2, and MCF7 with IC50 values of 16, 93, and 78 nM, respectively. SP 85 exhibited significantly (2-10 times) higher cytotoxicity against the tumor cell lines in comparison with HUVECs as the normal cell line, which also induced apoptosis in the tested cancerous cell line. This is the first report on the production of an "olivomycin A" derivative by a sponge-associated Streptomyces, showing the great potential of sponge-associated actinobacteria in producing cytotoxic natural products.

The redox couple avarol/avarone in the fight with malignant gliomas: the case study of U-251 MG cells.

This study aimed to screen in vitro antitumour activity of the redox couple avarol/avarone against the human malignant glioma cell line U-251 MG for the first time. Compared both with avarol and positive controls used (temozolomide and doxorubicin), avarone was found to be the most active compound with IC50 value below 1 µM (IC50 0.68 ± 0.04 µM, 96 h). Considerable less DNA damage in the cells treated with avarol and avarone vs. doxorubicin (105 & 123% vs. 299%, respectively; untreated U-251 MG cells were used as a control, 100%), coupled with no sign of cytotoxicity against the normal human foetal lung fibroblast MRC-5 cells (IC50 > 100 µM), has actually pointed out the importance of this marine sesquiterpenoid quinone structure as a promising lead compound in development of novel brain chemotherapeutics.

Selective Cytotoxicity of α-Santonin from the Persian Gulf Sponge Dysidea Avara on Pediatric ALL B-lymphocytes via Mitochondrial Targeting

Background: Acute lymphoblastic leukemia (ALL) is one of the most dominant malignancies among children, characterized by production of immature and dysfunctional blasts which are resistant to cytotoxic chemotherapeutic agents. Therefore, research protocols are currently focusing on discovery of novel anti-cancer agents to enhance survival rates and decrease unwanted side effects. Approximately two-thirds of the planet is covered by oceans with a massive range of marine organisms of interest to scientists in pharmaceutical fields. Methods: Among marine resources, sponges are known to have beneficial effects in the treatment of numerous malignancies. One fraction of crude extracts containing α-Santonin was made from the Persian Gulf marine sponge, Dysidea avara, and investigated for anticancer effects. Results: Treatment of ALL B-lymphocytes with the Dysidea avara extract caused augmentation in ROS generation, decline in mitochondrial membrane potential, mitochondrial swelling, release of cytochrome c from mitochondria and activation of caspase-3 only in mitochondria isolated from B-ALL lymphocytes. Conclusion: In brief, our results suggest that Dysidea avara extracts may selectively induce apoptosis in malignant pediatric lymphocytes.

In vitro evaluation of cytotoxic and mutagenic activity of avarol.

The cytotoxicity of avarol, a main secondary metabolite of the Mediterranean sponge Dysidea avara, was in vitro screened by MTT assay against four human tumour cell lines. The colon HT-29 tumour cells practically showed to be the only sensitive ones towards this organic compound. No toxicity was found against the fetal lung fibroblast MRC-5 cells at the concentrations tested. In comparison with doxorubicin, used as a positive control, avarol actually exhibited at least 588-fold less toxicity towards normal MRC-5 cells. Finally, comet assay indicated that DNA fragmentation was almost fivefold higher upon the treatment with doxorubicin, compared to avarol. The obtained results have actually confirmed that avarol scaffold may contribute to development of new cytostatics inspired by nature.

In vitro avarol does affect the growth of Candida sp.

This work extends in vitro screening of antimicrobial activity of avarol, the marine natural product firstly isolated from the Mediterranean sponge Dysidea avara. Its anticandidial activity was evaluated by microdilution method against eight Candida strains, two ATCC and six clinical ones. At a different extent this compound was proven to be active against all the strains tested (MIC 0.8-6.0 µg/mL and MFC 1.6-12.0 µg/mL, respectively). According to the best of our knowledge, this is the first report on avarol activity towards any yeast strain which may be of relevance for Alzheimer's disease. Indeed, avarol derivatives showing moderate AChE activity should be screened for anticandidial activity both in vitro and in vivo.