Targeting protein-protein interaction interfaces with antiviral N protein inhibitor in SARS-CoV-2.

Coronaviruses not only pose significant global public health threats but also cause extensive damage to livestock-based industries. Previous studies have shown that 5-benzyloxygramine (P3) targets the Middle East respiratory syndrome coronavirus (MERS-CoV) nucleocapsid (N) protein N-terminal domain (N-NTD), inducing non-native protein-protein interactions (PPIs) that impair N protein function. Moreover, P3 exhibits broad-spectrum antiviral activity against CoVs. The sequence similarity of N proteins is relatively low among CoVs, further exhibiting notable variations in the hydrophobic residue responsible for non-native PPIs in the N-NTD. Therefore, to ascertain the mechanism by which P3 demonstrates broad-spectrum anti-CoV activity, we determined the crystal structure of the SARS-CoV-2 N-NTD:P3 complex. We found that P3 was positioned in the dimeric N-NTD via hydrophobic contacts. Compared with the interfaces in MERS-CoV N-NTD, P3 had a reversed orientation in SARS-CoV-2 N-NTD. The Phe residue in the MERS-CoV N-NTD:P3 complex stabilized both P3 moieties. However, in the SARS-CoV-2 N-NTD:P3 complex, the Ile residue formed only one interaction with the P3 benzene ring. Moreover, the pocket in the SARS-CoV-2 N-NTD:P3 complex was more hydrophobic, favoring the insertion of the P3 benzene ring into the complex. Nevertheless, hydrophobic interactions remained the primary stabilizing force in both complexes. These findings suggested that despite the differences in the sequence, P3 can accommodate a hydrophobic pocket in N-NTD to mediate a non-native PPI, enabling its effectiveness against various CoVs.

Bioprospecting the American Alligator Peptidome for antiviral peptides against Venezuelan equine encephalitis virus.

The innate immune protection provided by cationic antimicrobial peptides (CAMPs) has been shown to extend to antiviral activity, with putative mechanisms of action including direct interaction with host cells or pathogen membranes. The lack of therapeutics available for the treatment of viruses such as Venezuelan equine encephalitis virus (VEEV) underscores the urgency of novel strategies for antiviral discovery. American alligator plasma has been shown to exhibit strong in vitro antibacterial activity, and functionalized hydrogel particles have been successfully employed for the identification of specific CAMPs from alligator plasma. Here, a novel bait strategy in which particles were encapsulated in membranes from either healthy or VEEV-infected cells was implemented to identify peptides preferentially targeting infected cells for subsequent evaluation of antiviral activity. Statistical analysis of peptide identification results was used to select five candidate peptides for testing, of which one exhibited a dose-dependent inhibition of VEEV and also significantly inhibited infectious titers. Results suggest our bioprospecting strategy provides a versatile platform that may be adapted for antiviral peptide identification from complex biological samples.

Protein Kinase C subtype δ interacts with Venezuelan equine encephalitis virus capsid protein and regulates viral RNA binding through modulation of capsid phosphorylation.

Protein phosphorylation plays an important role during the life cycle of many viruses. Venezuelan equine encephalitis virus (VEEV) capsid protein has recently been shown to be phosphorylated at four residues. Here those studies are extended to determine the kinase responsible for phosphorylation and the importance of capsid phosphorylation during the viral life cycle. Phosphorylation site prediction software suggests that Protein Kinase C (PKC) is responsible for phosphorylation of VEEV capsid. VEEV capsid co-immunoprecipitated with PKCδ, but not other PKC isoforms and siRNA knockdown of PKCδ caused a decrease in viral replication. Furthermore, knockdown of PKCδ by siRNA decreased capsid phosphorylation. A virus with capsid phosphorylation sites mutated to alanine (VEEV CPD) displayed a lower genomic copy to pfu ratio than the parental virus; suggesting more efficient viral assembly and more infectious particles being released. RNA:capsid binding was significantly increased in the mutant virus, confirming these results. Finally, VEEV CPD is attenuated in a mouse model of infection, with mice showing increased survival and decreased clinical signs as compared to mice infected with the parental virus. Collectively our data support a model in which PKCδ mediated capsid phosphorylation regulates viral RNA binding and assembly, significantly impacting viral pathogenesis.

(-)-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.

Crassolide Induces G2/M Cell Cycle Arrest, Apoptosis, and Autophagy in Human Lung Cancer Cells via ROS-Mediated ER Stress Pathways.

Crassolide, a cembranoid diterpene extracted from the soft coral Lobophytum crissum, has been proven to possess antioxidant and immunomodulatory properties. In the present study, we assessed the anticancer effects of crassolide on human H460 non-small-cell lung cancer (NSCLC) cells. We found that crassolide exerted cytotoxic effects on H460 cancer cells in vitro, inducing G2/M phase arrest and apoptosis. In addition, in H460 cells exposed to crassolide, the expression of the autophagy-related proteins LC3-II and beclin was increased, while the expression of p62 was decreased. Moreover, inhibiting autophagy with chloroquine (CQ) suppressed the crassolide-induced G2/M arrest and apoptosis of H460 cells. Moreover, we also found that crassolide induced endoplasmic reticulum (ER) stress in lung cancer cells by increasing the expression of ER stress marker proteins and that the crassolide-induced G2/M arrest, apoptosis, and autophagy were markedly attenuated by the ER stress inhibitor 4-phenylbutyric acid (4-PBA). Furthermore, we found that crassolide promoted reactive oxygen species (ROS) production by H460 cells and that the ROS inhibitor N-acetylcysteine (NAC) decreased the crassolide-induced ER stress, G2/M arrest, apoptosis, and autophagy. In conclusion, our findings show that crassolide inhibits NSCLC cell malignant biological behaviors for the first time, suggesting that this effect may be mechanistically achieved by inducing G2/M arrest, apoptosis, and autophagy through ROS accumulation, which activates the ER stress pathway. As a result of our findings, we now have a better understanding of the molecular mechanism underlying the anticancer effect of crassolide, and we believe crassolide might be a candidate for targeted cancer therapy.

Use of magnetic nanotrap particles in capturing Yersinia pestis virulence factors, nucleic acids and bacteria.

BACKGROUND: Many pathogens, including Yersinia pestis, cannot be consistently and reliably cultured from blood. New approaches are needed to facilitate the detection of proteins, nucleic acid and microorganisms in whole blood samples to improve downstream assay performance. Detection of biomarkers in whole blood is difficult due to the presence of host proteins that obscure standard detection mechanisms. Nanotrap® particles are micron-sized hydrogel structures containing a dye molecule as the affinity bait and used to detect host biomarkers, viral nucleic acids and proteins as well as some bacterial markers. Nanotraps have been shown to bind and enrich a wide variety of biomarkers and viruses in clinically relevant matrices such as urine and plasma. Our objective was to characterize the binding ability of Nanotrap particle type CN3080 to Y. pestis bacteria, bacterial proteins and nucleic acids from whole human blood in order to potentially improve detection and diagnosis. RESULTS: CN3080 Nanotraps bind tightly to Yersinia bacteria, even after washing, and we were able to visualize the co-localized Nanotraps and bacteria by electron microscopy. These magnetic hydrogel Nanotraps were able to bind Yersinia DNA, supporting the utility of Nanotraps for enhancing nucleic acid-based detection methods. Nanotraps were capable of increasing Y. pestis nucleic acid yield by fourfold from whole human blood compared to standard nucleic acid extraction. Interestingly, we found CN3080 Nanotraps to have a high affinity for multiple components of the Yersinia type III secretion system (T3SS), including chaperone proteins, Yop effector proteins and virulence factor protein LcrV (V). Using Nanotraps as a rapid upstream sample-prep tool, we were able to detect LcrV in human blood by western blotting with minimal blood interference in contrast to direct western blotting of blood samples in which LcrV was obscured. We were able to computationally model the interaction of LcrV with the CN3080 Nanotrap dye and found that it had a low delta-G, suggesting high affinity. Importantly, Nanotraps were also able to enhance detection of secreted Yersinia proteins by mass spectrometry. CONCLUSION: Upstream use of magnetic CN3080 Nanotrap particles may improve the downstream workflow though binding and enrichment of biomarkers and speed of processing. Utilization of Nanotrap particles can improve detection of Yersinia pestis proteins and nucleic acid from whole human blood and contribute to downstream assays and diagnostics including molecular methods such as sequencing and PCR and protein-based methods.

Kurarinone Attenuates Collagen-Induced Arthritis in Mice by Inhibiting Th1/Th17 Cell Responses and Oxidative Stress.

Kurarinone is a flavanone, extracted from Sophora flavescens Aiton, with multiple biological effects. Here, we determine the therapeutic potential of kurarinone and elucidate the interplay between kurarinone and the autoimmune disease rheumatoid arthritis (RA). Arthritis was recapitulated by induction of bovine collagen II (CII) in DBA/1 mice as a collagen-induced arthritis (CIA) model. After the establishment of the CIA, kurarinone was given orally from day 21 to 42 (100 mg/kg/day) followed by determination of the severity based on a symptom scoring scale and with histopathology. Levels of cytokines, anti-CII antibodies, and the proliferation and lineages of T cells from the draining lymph nodes were measured using ELISA and flow cytometry, respectively. The expressional changes, including STAT1, STAT3, Nrf2, KEAP-1, and heme oxygenase-1 (HO-1) changes in the paw tissues, were evaluated by Western blot assay. Oxidative stress featured with malondiadehyde (MDA) and hydrogen peroxide (H2O2) activities in paw tissues were also evaluated. Results showed that kurarinone treatment reduced arthritis severity of CIA mice, as well as their levels of proinflammatory cytokines, TNF-α, IL-6, IFN-γ, and IL-17A, in the serum and paw tissues. T cell proliferation was also reduced by kurarinone even under the stimulation of CII and anti-CD3 antibody. In addition, kurarinone reduced STAT1 and STAT3 phosphorylation and the proportions of Th1 and Th17 cells in lymph nodes. Moreover, kurarinone suppressed the production of MDA and H2O2. All while promoting enzymatic activities of key antioxidant enzymes, SOD and GSH-Px. In the paw tissues, upregulation of Nrf-2 and HO-1, and downregulation of KEAP-1 were observed. Overall, kurarinone showed an anti-inflammatory effect by inhibiting Th1 and Th17 cell differentiation and an antioxidant effect exerted in part through activating the Nrf-2/KEAP-1 pathway. These beneficial effects in CIA mice contributed to the amelioration of their arthritis, indicating that kurarinone might be an adjunct treatment option for rheumatoid arthritis.

The Gastroprotective Effect of Naringenin against Ethanol-Induced Gastric Ulcers in Mice through Inhibiting Oxidative and Inflammatory Responses.

Naringenin is a major flavanone found in grapes, tangelos, blood oranges, lemons, pummelo, and tangerines. It is known to have anti-inflammatory, antioxidant, anticancer, antimutagenic, antifibrogenic, and antiatherogenic pharmacological properties. This study aims to investigate the anti-inflammatory effects of naringenin in ethanol-induced gastric damage in vivo and ethanol-stimulated KATO III cells in vitro. Our results showed that pretreatment with naringenin significantly protected mice from ethanol-induced hemorrhagic damage, epithelial cell loss, and edema with leucocytes. It reduced gastric ulcers (GU) by suppressing ethanol-induced nuclear factor-κB (NF-κB) activity and decreasing the levels of nitric oxide (NO), malondialdehyde (MDA), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8), and myeloperoxidase (MPO). In addition, pretreatment with naringenin might inhibit the secretion of TNF-α, IL-6, and IL-8, as well as the proteins cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) via the suppression of NF-κB and mitogen-activated protein kinase (MAPK) signaling in ethanol-stimulated stomach epithelial KATO III cells. Together, the results of this study highlight the gastroprotective effect of naringenin in GU of mice by inhibiting gastric secretion and acidity, reducing inflammation and oxidative stress, suppressing NF-κB activity, and restoring the histological architecture. These findings suggested that naringenin has therapeutic potential in the alleviation of ethanol-induced GU.

Atractylodin Suppresses TGF-ß-Mediated Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells and Attenuates Bleomycin-Induced Pulmonary Fibrosis in Mice.

Idiopathic pulmonary fibrosis (IPF) is characterized by fibrotic change in alveolar epithelial cells and leads to the irreversible deterioration of pulmonary function. Transforming growth factor-beta 1 (TGF-ß1)-induced epithelial-mesenchymal transition (EMT) in type 2 lung epithelial cells contributes to excessive collagen deposition and plays an important role in IPF. Atractylodin (ATL) is a kind of herbal medicine that has been proven to protect intestinal inflammation and attenuate acute lung injury. Our study aimed to determine whether EMT played a crucial role in the pathogenesis of pulmonary fibrosis and whether EMT can be utilized as a therapeutic target by ATL treatment to mitigate IPF. To address this topic, we took two steps to investigate: 1. Utilization of anin vitro EMT model by treating alveolar epithelial cells (A549 cells) with TGF-ß1 followed by ATL treatment for elucidating the underlying pathways, including Smad2/3 hyperphosphorylation, mitogen-activated protein kinase (MAPK) pathway overexpression, Snail and Slug upregulation, and loss of E-cadherin. Utilization of an in vivo lung injury model by treating bleomycin on mice followed by ATL treatment to demonstrate the therapeutic effectiveness, such as, less collagen deposition and lower E-cadherin expression. In conclusion, ATL attenuates TGF-ß1-induced EMT in A549 cells and bleomycin-induced pulmonary fibrosis in mice.

Crassolide Suppresses Dendritic Cell Maturation and Attenuates Experimental Antiphospholipid Syndrome.

Antiphospholipid syndrome (APS) is an autoimmune disease characterized by the production of ß2-glycoprotein I (ß2GPI)-dependent autoantibodies, with vascular thrombosis or obstetrical complications. Around 20% of APS patients are refractory to current treatments. Crassolide, a cembranoid diterpene extracted from soft corals, is a potential therapeutic candidate. Here, to examine the anti-inflammatory properties of crassolide, we first determined its effects on bone marrow-derived and splenic dendritic cells (DC). Specifically, we applied lipopolysaccharide (LPS) or ß2GPI stimulation and measured the expressions of CD80 and CD86, and secretions of cytokines. We also determined in the OT-II mice, if bone marrow-derived DC was able to stimulate antigen-specific T cells. Moreover, we examined the therapeutic potential of crassolide postimmunization in a murine model of APS that depended on active immunization with ß2GPI. The vascular manifestations were evaluated in terms of fluorescein-induced thrombi in mesenteric microvessels, whereas the obstetric manifestations were evaluated based on the proportion of fetal loss after pregnancy. We also measured blood titers of anti-ß2GPI antibody, splenic cell proliferative responses and cytokine secretions after ß2GPI stimulation ex vivo. Finally, we determined in these mice, hematological, hepatic and renal toxicities of crassolide. Crassolide after LPS stimulation suppressed DC maturation and secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-12 and IL-23, and downstream T cell activation. Crassolide could partially ameliorate both the vascular and obstetric manifestations of APS in BALB/c mice. Both blood titers of anti-ß2GPI antibody and splenic cell proliferation after ß2GPI stimulation were reduced. Splenic Th1 and Th17 responses were also lowered after ß2GPI stimulation. Finally, within therapeutic doses of crassolide, we found no evidence of its toxicity. In conclusion, we showed the ability of crassolide to suppress DC and downstream T cell responses. Crassolide is therefore a potential candidate for adjunctive therapy in APS.

Alantolactone alleviates collagen-induced arthritis and inhibits Th17 cell differentiation through modulation of STAT3 signalling.

CONTEXT: Alantolactone, the bioactive component in Inula helenium L. (Asteraceae), exhibits multiple biological effects. OBJECTIVE: We aimed to determine the anti-inflammatory effect of alantolactone in a collagen-induced arthritis (CIA) mouse model and its immunomodulatory effects on Th17 differentiation. MATERIALS AND METHODS: A CIA mouse model was established with DBA/1 mice randomly divided into four groups (n = 6): healthy, vehicle and two alantolactone-treated groups (25 or 50 mg/kg), followed by oral administration of alantolactone to mice for 21 consecutive days after arthritis onset. The severity of CIA was evaluated by an arthritic scoring system and histopathological examination. Levels of cytokines and anti-CII antibodies as well as percentages of splenic Th17 and Th17 differentiation with or without alantolactone treatments (0.62, 1.2 or 2.5 µM) were detected with ELISA and flow cytometry, respectively. Western blot analysis was used to evaluate intracellular signalling in alantolactone-treated spleen cells. RESULTS: In CIA mice, alantolactone at 50 mg/kg attenuated RA symptoms, including high arthritis scores, infiltrating inflammatory cells, synovial hyperplasia, bone erosion and levels of the proinflammatory cytokines TNF-α, IL-6 and IL-17A, but not IL-10 in paw tissues. Alantolactone also reduced the number of splenic Th17 cells and the capability of naïve CD4+ T cells to differentiate into the Th17 subset by downregulating STAT3/RORγt signalling by as early as 24 h of treatment. DISCUSSION AND CONCLUSIONS: Alantolactone possesses an anti-inflammatory effect that suppresses murine CIA by inhibiting Th17 cell differentiation, suggesting alantolactone is an adjunctive therapeutic candidate to treat rheumatoid arthritis.

Sinensetin induces apoptosis and autophagy in the treatment of human T-cell lymphoma.

The present study was carried out to explore the effect of sinensetin in human T-cell lymphoma Jurkat cells and to reveal the underlying molecular mechanisms. We found that sinensetin significantly impeded Jurkat cell proliferation in a dose-dependent and time-dependent manner. Additionally, sinensetin treatment triggered apoptosis and autophagy in Jurkat cells. The apoptosis induction was related to a loss of mitochondrial membrane potential and to increased caspase-3/-8/-9 and poly(ADP-ribose) polymerase (PARP) cleavage. Sinensetin also induced autophagy, as evidenced by the formation of acidic vacuoles, the upregulation of LC3-II and beclin-1, and the downregulation of p62. In addition, the inhibition of autophagy by 3-methyladenine significantly enhanced the apoptosis rate and improved the sensitivity of the Jurkat cells to sinensetin. Moreover, sinensetin induced cell death, apoptosis, and autophagy through the activation of the reactive oxygen species/ c-Jun N-terminal kinase signaling pathway and the inhibition of the Akt/mTOR signaling pathways. In summary, our results revealed that sinensetin induced apoptosis and autophagy in human T-cell lymphoma Jurkat cells by activating reactive oxygen species/ c-Jun N-terminal kinase and blocking the Akt/mTOR signaling pathways. Thus, sinensetin might be a potential candidate in the development of antitumor drugs targeting T-cell leukemia.

Isorhamnetin ameliorates inflammatory responses and articular cartilage damage in the rats of monosodium iodoacetate-induced osteoarthritis.

Context: Osteoarthritis (OA) is a degenerative joint disease with damage to the articular cartilage. Active production of inflammatory cytokine/chemokine and matrix metalloproteinases may be found during the progression of OA. Isorhamnetin had the effects of anti-inflammatory, antioxidant, anti-ischemia, anti-atherosclerotic hepatoprotective and anticancer activities. Objective: Our study was focused on the effects of isorhamnetin treatment in OA. Materials and methods: We used monosodium iodoacetate (MIA)-induced OA rats to evaluate the effects of isorhamnetin related anti-inflammatory process. The rats in all groups were sacrificed on four weeks post-MIA injection. The measurements of knee joint swelling, histological analysis, serum inflammatory biomarkers and western blot were evaluated. Results: We found that isorhamnetin may reduce MIA-induced knee swelling by significantly reduction of articular cartilage damage.in rats. Suppression of pro-inflammatory cytokines production was found after isohamnetin treatment. Isorhamnetin inhibited the production of NO and PGE2, and the expression of iNOS and COX-2. The production of COMP, CTX-II and osteopontin (OPN) were also inhibited in MIA-induced OA rats. Discussion and conclusions: Isorhamnetin may modulate the inflammatory progression of OA in MIA-induced OA rats. The prevention of cartilage damage was found in OA after adequate isorhamnetin treatment. Isorhamnetin may serve as a potential agent for the management of OA.

Sclareol attenuates the development of atopic dermatitis induced by 2,4-dinitrochlorobenzene in mice.

Context: Atopic dermatitis is a common chronic inflammatory skin disease affecting up to 20% of children and 1% of adults worldwide. Treatment of atopic dermatitis include corticosteroids and immunosuppressants, such as calcineurin inhibitors and methotrexate. However, these treatments often bring about adverse effects including skin atrophy, osteoporosis, skin cancer, and metabolic syndrome. Objective: In this study, we evaluated the therapeutic effects and mechanisms of sclareol, a natural diterpene, on atopic dermatitis (AD)-like skin lesions induced by 2,4-dinitrochlorobenzene (DNCB) in mice. Materials and methods: To evaluate the effect of sclareol in vivo model, BALB/c mice were repeatedly injected intraperitoneally with sclareol (50 and 100 mg/kg) in 2,4-dinitrochlorobenzene (DNCB)-induced AD-like murine model. Major assays were enzyme-linked immunosorbent assay, histological analysis, flow cytometry, western blot analysis. Results: Intraperitoneal administration of sclareol (50 and 100 mg/kg) significantly attenuated AD-like symptoms, such as serum IgE levels, epidermal/dermal hyperplasia, and the numbers of infiltrated mast cells. In addition, systemic sclareol treatments reduced local pro-inflammatory cytokine concentrations, including IL-6, IL-1b, TNF-a, IL-4, IFN-g, and IL-17A, on AD-like lesions. Furthermore, we demonstrated that sclareol also suppressed T cell activation and the capability of cytokine productions (IFN-g, IL-4 and IL-17A) in response to DNCB stimulation. By examining the skin homogenate, we found that sclareol inhibited the AD-like severity likely through suppressions of both NF-kB translocation and phosphorylation of the MAP kinase pathway. Discussion and conclusions: Cumulatively, our results indicate that sclareol induced anti-inflammatory effects against the atopic dermatitis elicited by DNCB. Thus, sclareol is worth of being further evaluated for its potential therapeutic benefits for the clinical treatment of AD.

A Soft Coral-Derived Compound, 11-Dehydrosinulariolide, Induces G2/M Cell Cycle Arrest and Apoptosis in Small Cell Lung Cancer.

11-Dehydrosinulariolide, an active compound that is isolated from the cultured soft coral Sinularia flexibilis, has been suggested to show anti-tumor biological characteristics according to previous studies. However, its potential effect on small cell lung cancer (SCLC) remains unknown. The present study investigates the underlying mechanism for the treatment of SCLC in vitro and in vivo. Cell viability was examined using the methyl-thiazol-diphenyl-tetrazolium (MTT) assay. Flow cytometry was applied to evaluate cell cycle distribution and apoptosis. The expression of proteins related to the cell cycle and apoptosis was analyzed by Western blot analysis. Additionally, an in vivo study was performed to determine the anti-SCLC effect on an H1688 subcutaneous tumor in a BALB/c nude mouse model. 11-Dehydrosinulariolide inhibited cell growth, triggered G2/M arrest and induced H1688 cell apoptosis in a dose- and time-dependent manner. Additionally, 11-dehydrosinulariolide caused the accumulation of p53 and Bax, accompanied by the activation of DNA damage-inducing kinases, including ataxia-telangiectasia mutated (ATM) and checkpoint kinase 2 (CHK2). Moreover, 11-dehydrosinulariolide increased the activity of caspase-3 and -7, suggesting that caspases are involved in 11-dehydrosinulariolide-induced apoptosis. 11-Dehydrosinulariolide also increased the level of tumor suppressor phosphatase and tensin homolog (PTEN) and inhibited the expression of phosphorylated Akt. In the in vivo study, the intraperitoneal injection of 11-dehydrosinulariolide at a dosage of 10 mg/kg significantly inhibited tumor growth compared with the control treatment. Together, the data indicate that 11-dehydrosinulariolide induces G (2)/M cell cycle arrest and apoptosis through various cellular processes, including the upregulation of p53 and Bax, activation of ATM and Chk2, activation of caspase-3 and -7, and accumulation of PTEN, leading to inhibition of the Akt pathway. These findings suggest that 11-dehydrosinulariolide might serve as a promising chemotherapy drug in the treatment of SCLC.

Therapeutic Potential of Sclareol in Experimental Models of Rheumatoid Arthritis.

Previous studies have shown that the natural diterpene compound, sclareol, potentially inhibits inflammation, but it has not yet been determined whether sclareol can alleviate inflammation associated with rheumatoid arthritis (RA). Here, we utilized human synovial cell line, SW982, and an experimental murine model of rheumatoid arthritis, collagen-induced arthritis (CIA), to evaluate the therapeutic effects of sclareol in RA. Arthritic DBA/1J mice were dosed with 5 and 10 mg/kg sclareol intraperitoneally every other day over 21 days. Arthritic severity was evaluated by levels of anti-collagen II (anti-CII) antibody, inflammatory cytokines, and histopathologic examination of knee joint tissues. Our results reveal that the serum anti-CII antibody, cytokines interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and IL-17, as well as Th17 and Th1 cell population in inguinal lymph nodes, were significantly lower in sclareol-treated mice compared to the control group. Also, the sclareol treatment groups showed reduced swelling in the paws and lower histological arthritic scores, indicating that sclareol potentially mitigates collagen-induced arthritis. Furthermore, IL-1β-stimulated SW982 cells secreted less inflammatory cytokines (TNF-α and IL-6), which is associated with the downregulation of p38-mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and NF-κB pathways. Overall, we demonstrate that sclareol could relieve arthritic severities by modulating excessive inflammation and our study merits the pharmaceutical development of sclareol as a therapeutic treatment for inflammation associated with RA.

Effective inhibition of MERS-CoV infection by resveratrol.

BACKGROUND: Middle East Respiratory Syndrome coronavirus (MERS-CoV) is an emerging viral pathogen that causes severe morbidity and mortality. Up to date, there is no approved or licensed vaccine or antiviral medicines can be used to treat MERS-CoV-infected patients. Here, we analyzed the antiviral activities of resveratrol, a natural compound found in grape seeds and skin and in red wine, against MERS-CoV infection. METHODS: We performed MTT and neutral red uptake assays to assess the survival rates of MERS-infected Vero E6 cells. In addition, quantitative PCR, western blotting, and immunofluorescent assays determined the intracellular viral RNA and protein expression. For viral productivity, we utilized plaque assays to confirm the antiviral properties of resveratrol against MERS-CoV. RESULTS: Resveratrol significantly inhibited MERS-CoV infection and prolonged cellular survival after virus infection. We also found that the expression of nucleocapsid (N) protein essential for MERS-CoV replication was decreased after resveratrol treatment. Furthermore, resveratrol down-regulated the apoptosis induced by MERS-CoV in vitro. By consecutive administration of resveratrol, we were able to reduce the concentration of resveratrol while achieving inhibitory effectiveness against MERS-CoV. CONCLUSION: In this study, we first demonstrated that resveratrol is a potent anti-MERS agent in vitro. We perceive that resveratrol can be a potential antiviral agent against MERS-CoV infection in the near future.

Sinularin induces DNA damage, G2/M phase arrest, and apoptosis in human hepatocellular carcinoma cells.

BACKGROUND: Sinularin isolated from the cultured soft coral Sinularia flexibilis has been reported to exert potent cytotoxic effects against particular types of cancer. This study was carried out to investigate the cytotoxic effects in sinularin-treated human hepatocellular carcinoma cells, HepG2, and to subsequently explore the underlying molecular mechanisms. METHODS: TheMTT (3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyl- tetrazolium bromide) method was used to evaluate the cytotoxicity of sinularin on HepG2 and Hep3B cell lines. Furthermore, the cell cycle distribution assay, apoptosis assay, and western blot analysis in vitro were used to explore the possible mechanisms of action. RESULTS: From the results of our study, cell viability was obviously inhibited by sinularin in a dose-dependent manner. In addition, our results suggested that sinularin triggered DNA damage and subsequently induced cell cycle G2/M arrest associated with up-regulation of p-ATM (Ser(1981)), p-Chk2 (Tyr(68)), p-cdc2 (Tyr(15)), and p53 coupled with increased expression of downstream proteins p21 and down-regulation of p-cdc25 (Ser(216)). Moreover, the results of the apoptosis assay and western blot analysis indicated that the cytotoxic activity could be related to mitochondrial apoptosis, characterized by decrease of Bcl-2 expression, disruption of mitochondrial membrane potential, and sequential activation of caspases and Poly (ADP-ribose) polymerase (PARP). CONCLUSIONS: This study reveals for the first time the anti-HCC activities of sinularin, the active compound isolated from the cultured soft coral Sinularia flexibilis. We believe that our results warrant further evaluation of sinularin as a new anti-HCC chemotherapeutic agent.

RK-33, a small molecule inhibitor of host RNA helicase DDX3, suppresses multiple variants of SARS-CoV-2.

SARS-CoV-2, the virus behind the deadly COVID-19 pandemic, continues to spread globally even as vaccine strategies are proving effective in preventing hospitalizations and deaths. However, evolving variants of the virus appear to be more transmissive and vaccine efficacy towards them is waning. As a result, SARS-CoV-2 will continue to have a deadly impact on public health into the foreseeable future. One strategy to bypass the continuing problem of newer variants is to target host proteins required for viral replication. We have used this host-targeted antiviral (HTA) strategy that targets DDX3, a host DEAD-box RNA helicase that is usurped by SARS-CoV-2 for virus production. We demonstrated that targeting DDX3 with RK-33, a small molecule inhibitor, reduced the viral load in four isolates of SARS-CoV-2 (Lineage A, and Lineage B Alpha, Beta, and Delta variants) by one to three log orders in Calu-3 cells. Furthermore, proteomics and RNA-seq analyses indicated that most SARS-CoV-2 genes were downregulated by RK-33 treatment. Also, we show that the use of RK-33 decreases TMPRSS2 expression, which may be due to DDX3s ability to unwind G-quadraplex structures present in the TMPRSS2 promoter. The data presented supports the use of RK-33 as an HTA strategy to control SARS-CoV-2 infection, irrespective of its mutational status, in humans.

RK-33, a small molecule inhibitor of host RNA helicase DDX3, suppresses multiple variants of SARS-CoV-2.

SARS-CoV-2, the virus behind the deadly COVID-19 pandemic, continues to spread globally even as vaccine strategies are proving effective in preventing hospitalizations and deaths. However, evolving variants of the virus appear to be more transmissive and vaccine efficacy toward them is waning. As a result, SARS-CoV-2 will continue to have a deadly impact on public health into the foreseeable future. One strategy to bypass the continuing problem of newer variants is to target host proteins required for viral replication. We have used this host-targeted antiviral (HTA) strategy that targets DDX3X (DDX3), a host DEAD-box RNA helicase that is usurped by SARS-CoV-2 for virus production. We demonstrated that targeting DDX3 with RK-33, a small molecule inhibitor, reduced the viral load in four isolates of SARS-CoV-2 (Lineage A, and Lineage B Alpha, Beta, and Delta variants) by one to three log orders in Calu-3 cells. Furthermore, proteomics and RNA-seq analyses indicated that most SARS-CoV-2 genes were downregulated by RK-33 treatment. Also, we show that the use of RK-33 decreases TMPRSS2 expression, which may be due to DDX3s ability to unwind G-quadraplex structures present in the TMPRSS2 promoter. The data presented support the use of RK-33 as an HTA strategy to control SARS-CoV-2 infection, irrespective of its mutational status, in humans.