Demethoxymurrapanine, an indole-naphthoquinone alkaloid, inhibits the proliferation of oral cancer cells without major side effects on normal cells.

Antioral cancer drugs need a greater antiproliferative impact on cancer than on normal cells. Demethoxymurrapanine (DEMU) inhibits proliferation in several cancer cells, but an in-depth investigation was necessary. This study evaluated the proliferation-modulating effects of DEMU, focusing on oral cancer and normal cells. DEMU (0, 2, 3, and 4 µg/mL) at 48 h treatments inhibited the proliferation of oral cancer cells (the cell viability (%) for Ca9-22 cells was 100.0 ± 2.2, 75.4 ± 5.6, 26.0 ± 3.8, and 15.4 ± 1.4, and for CAL 27 cells was 100.0 ± 9.4, 77.2 ± 5.9, 57.4 ± 10.7, and 27.1 ± 1.1) more strongly than that of normal cells (the cell viability (%) for S-G cells was 100.0 ± 6.6, 91.0 ± 4.6, 95.0 ± 2.6, and 95.8 ± 5.5), although this was blocked by the antioxidant N-acetylcysteine. The presence of oxidative stress was evidenced by the increase of reactive oxygen species and mitochondrial superoxide and the downregulation of the cellular antioxidant glutathione in oral cancer cells, but these changes were minor in normal cells. DEMU also caused greater induction of the subG1 phase, extrinsic and intrinsic apoptosis (annexin V and caspases 3, 8, and 9), and DNA damage (γH2AX and 8-hydroxy-2-deoxyguanosine) in oral cancer than in normal cells. N-acetylcysteine attenuated all these DEMU-induced changes. Together, these data demonstrate the preferential antiproliferative function of DEMU in oral cancer cells, with the preferential induction of oxidative stress, apoptosis, and DNA damage in these cancer cells, and low cytotoxicity toward normal cells.

Scalarane-Type Sesterterpenoids from the Marine Sponge Lendenfeldia sp. Alleviate Inflammation in Human Neutrophils.

Sponge-derived scalaranes are remarkable sesterterpenoids previously found to exhibit profound inhibitory effects against neutrophilic inflammation. In our current work, we constructed the metabolomic profile of marine sponge Lendenfeldia sp. for the first time using a tandem mass spectrometry (MS/MS) molecular networking approach. The results highlighted the rich chemical diversity of these scalaranes, motivating us to conduct further research to discover novel scalaranes targeting neutrophilic inflammation. MS- and NMR-assisted isolation and elucidation led to the discovery of seven new homoscalaranes, lendenfeldaranes K-Q (1-7), characterized by methylation at C-24, together with five known derivatives, lendenfeldarane B (8), 25-nor-24-methyl-12,24-dioxoscalar-16-en-22-oic acid (9), 24-methyl-12,24,25-trioxoscalar-16-en-22-oic acid (10), felixin B (11), and 23-hydroxy-20-methyldeoxoscalarin (12). Scalaranes 1-4 and 6-12 were assayed against superoxide anion generation and elastase release, which represented the neutrophilic inflammatory responses of respiratory burst and degranulation, respectively. The results indicated that 1-3 and 6-12 exhibited potential anti-inflammatory activities (IC50 for superoxide anion scavenging: 0.87~6.57 µM; IC50 for elastase release: 1.12~6.97 µM).

Sponge-Derived 24-Homoscalaranes as Potent Anti-Inflammatory Agents.

Scalarane-type sesterterpenoids are known for their therapeutic potential in cancer treatments. However, the anti-inflammatory properties of this class of metabolites remain elusive. Our current work aimed to investigate the anti-inflammatory scalaranes from marine sponge Lendenfeldia sp., resulting in the isolation of six new 24-homoscalaranes, lendenfeldaranes E-J (1-6). The structures of the new metabolites were determined by extensive spectroscopic analyses, and the absolute configuration of 1 was established by electronic circular dichroism (ECD) calculations. Compounds 2 and 3 were discovered to individually reduce the generation of superoxide anions, and compound 1 displayed an inhibitor effect on the release of elastase. These three compounds were proven to be the first anti-neutrophilic scalaranes.

Ubiquitin-Proteasome Modulating Dolabellanes and Secosteroids from Soft Coral Clavularia flava.

We performed a high-content screening (HCS) assay aiming to discover bioactive molecules with proteasome inhibitory activity. By structural elucidation, we identified six compounds purified from soft coral Clavularia flava, which potentiates proteasome inhibition. Chemical structure elucidation revealed they are dolabellane- and secosteroid-based compounds including a new dolabellane, clavinflol C (1), three known dolabellanes, stolonidiol (2), stolonidiol-17-acetate (3), and clavinflol B (4) as well as two new secosteroids, 3,11-dihydroxy-24-methyl-9,11-secocholest-5-en-9,23-dione (5) and 3,11-dihydroxy-24-methylene-9,11-secocholest-5-en-9,23-dione (6). All six compounds show less cytotoxicity than those of known proteasome inhibitors, bortezomib and MG132. In summary, the high-content measurements of control inhibitors, bortezomib and MG132, manifest the highest ratio >2 in high-content measurement. Of the isolated compounds, 2 and 5 showed higher activity, followed by 3 and 6, and then 1 and 4 exhibited moderate inhibition.

Probing Anti-Proliferative 24-Homoscalaranes from a Sponge Lendenfeldiasp.

In the current study, an NMR spectroscopic pattern-based procedure for probing scalarane derivatives was performed and four new 24-homoscalaranes, lendenfeldaranes A-D (1- 4), along with three known compounds, 12α-acetoxy-22-hydroxy-24-methyl-24-oxoscalar-16-en- 25-al (5), felixin F (6), and 24-methyl-12,24,25-trioxoscalar-16-en-22-oic acid (7) were isolated from the sponge Lendenfeldia sp. The structures of scalaranes 1-7 were elucidated on the basis of spectroscopic analysis. Scalaranes 1-7 were further evaluated for their cytotoxicity toward a series of human cancer cell lines and the results suggested that 5 and 7 dominated in the anti- proliferative activity of the extract. The 18-aldehyde functionality was found to play a key role in their activity.

Lemnalol Modulates the Electrophysiological Characteristics and Calcium Homeostasis of Atrial Myocytes.

Sepsis, an inflammatory response to infection provoked by lipopolysaccharide (LPS), is associated with high mortality, as well as ischemic stroke and new-onset atrial arrhythmia. Severe bacterial infections causing sepsis always result in profound physiological changes, including fever, hypotension, arrhythmia, necrosis of tissue, systemic multi-organ dysfunction and finally death. LPS challenge-induced inflammatory responses during sepsis may increase the likelihood of the arrhythmogenesis. Lemnalol is known to possess potent anti-inflammatory effects. This study examined whether Lemnalol (0.1 µM) could modulate the electrophysiological characteristics and calcium homeostasis of atrial myocytes under the influence of LPS (1µg/mL). Under challenge with LPS, Lemnalol-treated LA myocytes, had a longer AP duration at 20%, 50% and 90% repolarization of the amplitude, compared to the LPS-treated cells. LPS-challenged LA myocytes showed increased late sodium current, Na+-Ca2+ exchanger current, transient outward current, rapid component of delayed rectifier potassium current, tumor necrosis factor-α, NF-κB and increased phosphorylation of ryanodine receptor (RyR), but a lower L-type Ca2+ current than the control LA myocytes. Exposure to Lemnalol reversed the LPS-induced effects. The LPS-treated and control groups of LA myocytes, with or without the existence of Lemnalol. showed no apparent alterations in the sodium current amplitude or Cav1.2 expression. The expression of sarcoendoplasmic reticulum calcium transport ATPase (SERCA2) was reduced by LPS treatment, while Lemnalol ameliorated the LPS-induced alterations. The phosphorylation of RyR was enhanced by LPS treatment, while Lemnalol attenuated the LPS-induced alterations. In conclusion, Lemnalol modulates LPS-induced alterations of LA calcium homeostasis and blocks the NF-κB pathways, which may contribute to the attenuation of LPS-induced arrhythmogenesis.

A High-Content Screening Assay for the Discovery of Novel Proteasome Inhibitors from Formosan Soft Corals.

The ubiquitin-proteasome system (UPS) is a major proteolytic pathway that safeguards protein homeostasis. The main 26S proteasome consists of a 20S catalytic core proteasome and a 19S substrate recognition proteasome. UPS dysfunction underlies many important clinical diseases involving inflammation, tumors, and neurodegeneration. Currently, three 20S proteasome inhibitors, bortezomib, carfilzomib, and ixazomib, have been approved for the treatment of multiple myeloma. We aim to screen UPS inhibitors for biomedical purposes. The protein interaction network of human cytomegalovirus UL76 targets UPS, resulting in aggregations of ubiquitinated proteins termed aggresomes. In this study, we demonstrated that cell-based high-content measurements of EGFP-UL76 aggresomes responded to bortezomib and MG132 treatment in a dose-dependent manner. Employing this high-content screening (HCS) assay, we screened natural compounds purified from Formosan soft corals. Four cembrane-based compounds, sarcophytonin A (1), sarcophytoxide (2), sarcophine (3), and laevigatol A (4), were found to enhance the high-content profiles of EGFP-UL76 aggresomes with relative ratios of 0.2. By comparison to the mechanistic action of proteasome inhibitors, compounds 1 and 3 modulated the accumulation of ubiquitinated proteins, with a unique pattern likely targeting 19S proteasome. We confirmed that the EGFP-UL76 aggresome-based HCS system greatly improves the efficacy and sensitivity of the identification of proteasome inhibitors.

Xeniaphyllane-Derived Terpenoids from Soft Coral Sinularia nanolobata.

A novel tetranorditerpenoid, sinubatin A (1) (having an unprecedented carbon skeleton), a new norditerpenoid, sinubatin B (2) (a 4,5-epoxycaryophyllene possessing an unusual methylfuran moiety side chain), and a known diterpenoid, gibberosin J (3) were isolated from soft coral Sinulariananolobata. The structures of the new compounds were elucidated by extensive analysis of spectroscopic data.

New Cytotoxic Terpenoids from Soft Corals Nephthea chabroli and Paralemnalia thyrsoides.

A novel cytotoxic diterpenoid, chabrolin A (1) (possessing an unprecedented terpenoid skeleton), as well as three new cytotoxic sesquiterpenoids, parathyrsoidins E-G (2-4), were isolated by cytotoxicity-guided fractionation from soft corals Nephtheachabroli and Paralemnalia thyrsoides. The structures of the new compounds were determined by extensive analysis of spectroscopic data.

Coral-Derived Natural Marine Compound GB9 Impairs Vascular Development in Zebrafish.

Blood vessels in vertebrates are established and genetically controlled in an evolutionarily-conserved manner during embryogenesis. Disruption of vascular growth by chemical compounds or environmental hormones may cause developmental defects. This study analyzed the vascular impacts of marine compound GB9 in zebrafish. GB9 was isolated from the marine soft coral Capnella imbricata and had shown anti-neuroinflammatory and anti-nociceptive activities. However, the role of GB9 on vascular development has not been reported. We first tested the survival rate of embryos under exogenous 5, 7.5, 10, and 15 µM GB9 added to the medium and determined a sub-lethal dosage of 10 µM GB9 for further assay. Using transgenic Tg(fli:eGFP) fish to examine vascular development, we found that GB9 treatment impaired intersegmental vessel (ISV) growth and caudal vein plexus (CVP) patterning at 25 hours post-fertilization (hpf) and 30 hpf. GB9 exposure caused pericardial edema and impaired circulation at 48-52 hpf, which are common secondary effects of vascular defects and suggest the effects of GB9 on vascular development. Apoptic cell death analysis showed that vascular defects were not caused by cell death, but were likely due to the inhibition of migration and/or proliferation by examining ISV cell numbers. To test the molecular mechanisms of vascular defects in GB9-treated embryos, we examined the expression of vascular markers and found the decreased expression of vascular specific markers ephrinb2, flk, mrc1, and stabilin. In addition, we examined whether GB9 treatment impairs vascular growth due to an imbalance of redox homeostasis. We found an enhanced effect of vascular defects during GB9 and H2O2 co-treatment. Moreover, exogenous N-acetyl-cysteine (NAC) treatment rescued the vascular defects in GB9 treated embryos. Our results showed that GB9 exposure causes vascular defects likely mediated by the imbalance of redox homeostasis.

Mollisolactones A and B, novel dinormonoterpenes from the soft coral Sinularia mollis.

Two novel dinormonoterpenes, designated as mollisolactones A and B, were discovered from the soft coral Sinularia mollis on the basis of a chromatographic and NMR spectroscopy-based fractionation. Their structures were solved through analysis of comprehensive 1D and 2D NMR spectroscopic data and HRESIMS experiments. The biological activities of the obtained metabolites were evaluated for cytotoxicity against A-459 (human lung carcinoma), HT-29 (human colon adenocarcinoma), and P-388 (mouse lymphocytic leukemia) cancer cell lines as well as antiviral activity against HCMV (human cytomegalovirus).

Anti-Inflammatory and Analgesic Effects of the Marine-Derived Compound Excavatolide B Isolated from the Culture-Type Formosan Gorgonian Briareum excavatum.

In recent years, several marine-derived compounds have been clinically evaluated. Diterpenes are secondary metabolites from soft coral that exhibit anti-inflammatory, anti-tumor and cytotoxic activities. In the present study, we isolated a natural diterpene product, excavatolide B, from cultured Formosan gorgonian Briareum excavatum and investigated its anti-inflammatory activities. We found that excavatolide B significantly inhibited the mRNA expression of the proinflammatory mediators, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in lipopolysaccharide (LPS)-challenged murine macrophages (RAW 264.7). We also examined the anti-inflammatory and anti-nociceptive effects of excavatolide B on intraplantar carrageenan-induced inflammatory responses. Excavatolide B was found to significantly attenuate carrageenan-induced nociceptive behaviors, mechanical allodynia, thermal hyperalgesia, weight bearing deficits and paw edema. In addition, excavatolide B inhibited iNOS, as well as the infiltration of immune cells in carrageenan-induced inflammatory paw tissue.

Sandensone A, a novel sesquiterpenoid from the Formosan soft coral Sinularia sandensis.

A chromatographic and NMR spectroscopy-based fractionation on the acetone extracts of the soft coral Sinularia sandensis led to the isolation of a novel sesquiterpenoid, sandensone A (1). The structure of 1 was elucidated based on comprehensive 1D and 2D NMR spectroscopic data as well as HRESIMS spectrometry. The absolute configuration at C-12 of 1 was determined as R using a modified Mosher's method. The cytotoxicity against A549 (human lung carcinoma), HT-29 (human colon adenocarcinoma), and P-388 (mouse lymphocytic leukemia) cancer cell lines as well as antiviral activity against HCMV (human cytomegalovirus) were evaluated in vitro.

Polyoxygenated cembrane diterpenoids from the soft coral Sarcophyton ehrenbergi.

Five new polyoxygenated cembranoids, named (+)-1,15-epoxy-2-methoxy-12-methoxycarbonyl-11E-sarcophytoxide (1), (+)-2-epi-12-methoxycarbonyl-11E-sarcophine (2), 3,4-epoxyehrenberoxide A (3), ehrenbergol D (4) and ehrenbergol E (5), were obtained from the soft coral Sarcophyton ehrenbergi. The structures of 1-5 were established on the basis of comprehensive NMR and HR-ESI-MS analyses and by comparison with reported data in the literature. Compounds 4 and 5 showed moderate cytotoxicity against P-388 (mouse lymphocytic leukemia) cancer cell line with EC50 values of 2.0 and 3.0 µM, respectively. Compound 2 exhibited slight antiviral activity against HCMV (human cytomegalovirus) with IC50 values of 25.0 µg/mL.

Calpains mediate the proteolytic modification of human cytomegalovirus UL112-113 proteins.

The human cytomegalovirus (HCMV) UL112-113 gene is implicated in lytic viral replication. The UL112-113 proteins p34, p43, p50 and p84 are expressed via alternative splicing. However, the mechanism for the generation of three additional virus-associated proteins (p20, p26 and p28), which share the UL112 reading frame, remains unknown. Bioinformatic analyses indicated that p34, p43, p50 and p84 contain potential PEST-like degradation motifs. In this study, inhibitors of calpains, lysosomes and proteasomes reduced p20, p26 and p28 levels in virus-infected cells, suggesting the involvement of proteolytic modification. Moreover, maitotoxin, which increases intracellular calcium levels and activates calpain activity, induced the intracellular proteolysis of p34 into p20, p26 and p28 and the cleavage of p43, p50 and p84 into p38 and a novel protein, p34c. Proteolytic assays further indicated that p34, p43, p50 and p84 were substrates of calpain-1 and calpain-2 and that they generated proteolytic products that corresponded to those detected during the HCMV infectious period. Furthermore, substitution mutations in the putative calpain cleavage sites of p34 reduced accumulation of proteolytic products. The knockdown of endogenous calpain-1 and calpain-2 by RNA interference reduced accumulation of p20, p26 and p28 and concurrently increased levels of nascent p43, p50 and p84 during the infectious cycle. Intriguingly, calpain depletion enhanced viral genome synthesis. Moreover, HCMV-permissive cells that stably expressed p20, p26 or p28 exhibited reduced viral genome synthesis and mature virus production. Our findings suggest that cognate UL112-113 proteins derived from calpain-catalysed proteolysis are involved in the HCMV replication process.

Lemnalol attenuates mast cell activation and osteoclast activity in a gouty arthritis model.

OBJECTIVES: In this study, we investigated the effects of a soft coral-derived anti-inflammatory compound, lemnalol, on mast cell (MC) function and osteoclast activity in rats with monosodium urate (MSU) crystal-induced gouty arthritis. METHODS: In this study, we examined the therapeutic effects of lemnalol on intra-articular injection of MSU induces gouty arthritis with the measurement of ankle oedema. Toluidine blue staining were used to analyse the infiltration and the percentage degranulation MCs. Immunohistochemical analysis showed CD117, transforming growth factor beta 1 (TGF-ß1), matrix metalloproteinase 9 (MMP-9), the osteoclast markers cathepsin K and tartrate-resistant acid phosphatase (TRAP) protein expression in ankle tissue. KEY FINDINGS: We found that both infiltration and degranulation of MCs increased at 24 h after MSU injection in the ankle joint. Immunohistochemical analysis showed that MSU induced upregulation of TGF-ß1, MMP-9, the osteoclast markers cathepsin K and TRAP in ankle tissues. Administration of lemnalol ameliorated MSU-induced TGF-ß1, MMP-9, cathepsin K and TRAP protein expression. CONCLUSIONS: Taken together, our results show that MSU-induced gouty arthritis is accompanied by osteoclast-related protein upregulation and that lemnalol treatment may be beneficial for the attenuation of MC infiltration and degranulation and for suppressing osteoclast activation in gouty arthritis.

Secocrassumol, a seco-cembranoid from the Dongsha Atoll soft coral Lobophytum crassum.

Chemical investigations on the Dongsha Atoll soft coral Lobophytum crassum led to the purification of a new seco-cembranoid, secocrassumol. The structural elucidation was established by extensive NMR, HRESIMS and CD data. The absolute configuration at C-12 was determined as S using a modified Mosher's acylation. Secocrassumol differs from previously known marine seco-cembranoid in that it possesses an unprecedented skeleton functionalized at C11-C12 bond cleavage. Secocrassumol showed antiviral activity against human cytomegalovirus (HCMV) with an IC50 value of 5.0 µg/mL.

Numerosol A-D, new cembranoid diterpenes from the soft coral Sinularia numerosa.

Four new cembrane-type diterpenes; numerosol A-D (1-4); along with a known steroid; gibberoketosterol (5); were isolated from the Taiwanese soft coral Sinularia numerosa. The structures of these metabolites were determined by extensive analysis of spectroscopic data. Gibberoketosterol (5) exhibited cytotoxicity against P-388 (mouse lymphocytic leukemia) cell line with an ED50 of 6.9 µM.

Sinugyrosanolide A, an unprecedented C-4 norcembranoid, from the Formosan soft coral Sinularia gyrosa.

Chemical investigations on the acetone extract of the Formosan soft coral Sinularia gyrosa have obtained a novel C-4 norcembranoid possessing an unprecedented tricyclo[9.3.0.0(3,8)]tetradecane skeleton, namely sinugyrosanolide A. The NMR spectroscopic data of the novel norcembranoid were completely assigned by using a combination of 2D NMR experiments including (1)H-(1)H COSY, HSQC, HMBC, and NOESY. The cytotoxicities, anti-HCMV (human cytomegalovirus) endonuclease activities and antibacterial activities were evaluated in vitro. It showed moderate cytotoxicity against P-388 (mouse lymphocytic leukemia) cancer cell line with an EC50 of 11.8µM.

Kelsoenethiol and dikelsoenyl ether, two unique kelsoane-type sesquiterpenes, from the Formosan soft coral Nephthea erecta.

Two new kelsoane-type sesquiterpenes, namely kelsoenethiol (1) and dikelsoenyl ether (2), were obtained from the Formosan soft coral Nephthea erecta. Their structures were elucidated through extensive spectroscopic analyses, ESI orbitrap mass and quantum chemical calculations (QCC). The cytotoxicity against A-459 (human lung carcinoma), P-388 (mouse lymphocytic leukemia), and HT-29 (human colon adenocarcinoma) cancer cell lines of 1 and 2 was evaluated in vitro. Compound 1 showed cytotoxicity against P-388 and HT-29 cells with ED50s of 1.3 and 1.8 µg/mL, respectively.