CDH17 nanobodies facilitate rapid imaging of gastric cancer and efficient delivery of immunotoxin.

BACKGROUND: It is highly desirable to develop new therapeutic strategies for gastric cancer given the low survival rate despite improvement in the past decades. Cadherin 17 (CDH17) is a membrane protein highly expressed in cancers of digestive system. Nanobody represents a novel antibody format for cancer targeted imaging and drug delivery. Nanobody targeting CHD17 as an imaging probe and a delivery vehicle of toxin remains to be explored for its theragnostic potential in gastric cancer. METHODS: Naïve nanobody phage library was screened against CDH17 Domain 1-3 and identified nanobodies were extensively characterized with various assays. Nanobodies labeled with imaging probe were tested in vitro and in vivo for gastric cancer detection. A CDH17 Nanobody fused with toxin PE38 was evaluated for gastric cancer inhibition in vitro and in vivo. RESULTS: Two nanobodies (A1 and E8) against human CDH17 with high affinity and high specificity were successfully obtained. These nanobodies could specifically bind to CDH17 protein and CDH17-positive gastric cancer cells. E8 nanobody as a lead was extensively determined for tumor imaging and drug delivery. It could efficiently co-localize with CDH17-positive gastric cancer cells in zebrafish embryos and rapidly visualize the tumor mass in mice within 3 h when conjugated with imaging dyes. E8 nanobody fused with toxin PE38 showed excellent anti-tumor effect and remarkably improved the mice survival in cell-derived (CDX) and patient-derived xenograft (PDX) models. The immunotoxin also enhanced the anti-tumor effect of clinical drug 5-Fluorouracil. CONCLUSIONS: The study presents a novel imaging and drug delivery strategy by targeting CDH17. CDH17 nanobody-based immunotoxin is potentially a promising therapeutic modality for clinical translation against gastric cancer.

Recent advances in mesoporous silica nanoparticle-based targeted drug-delivery systems for cancer therapy.

Targeted drug-delivery systems are a growing research topic in tumor treatment. In recent years, mesoporous silica nanoparticles (MSNs) have been extensively studied and applied in noninvasive and biocompatible drug-delivery systems for tumor therapy due to their outstanding advantages, which include high surface area, large pore volume, tunable pore size, easy surface modification and stable framework. The advances in the application of MSNs for anticancer drug targeting are covered and highlighted in this review, and the challenges and prospects of MSN-based targeted drug-delivery systems are discussed. This review provides new insights for researchers interested in targeted drug-delivery systems against cancer.

Heat Shock Protein A6 Is Especially Involved in Enterovirus 71 Infection.

Hand foot and mouth disease (HFMD) caused by Enterovirus 71 (EV71) infection is still a major infectious disease threatening children's life and health in the absence of effective antiviral drugs due to its high prevalence and neurovirulence. A study of EV71-specific host response might shed some light on the reason behind its unique epidemiologic features and help to find means to conquer EV71 infection. We reported that host heat shock protein A6 (HSPA6) was induced by EV71 infection and involved infection in both Rhabdomyosarcoma (RD) cells and neurogliocytes. Most importantly, we found that EV71 did not induce the expression of other heat shock proteins HSPA1, HSPA8, and HSPB1 under the same conditions, and other HFMD-associated viruses including CVA16, CVA6, CVA10, and CVB1-3 did not induce the upregulation of HSPA6. In addition, EV71 infection enhanced the cytoplasmic aggregation of HSPA6 and its colocalization with viral capsid protein VP1. These findings suggest that HSPA6 is a potential EV71-specific host factor worthy of further study.

The Antiviral Effects of Jasminin via Endogenous TNF-α and the Underlying TNF-α-Inducing Action.

Previous studies have reported that recombinant tumor necrosis factor (TNF)-α has powerful antiviral activity but severe systematic side effects. Jasminin is a common bioactive component found in Chinese herbal medicine beverage "Jasmine Tea". Here, we report that jasminin-induced endogenous TNF-α showed antiviral activity in vitro. The underlying TNF-α-inducing action of jasminin was also investigated in RAW264.7 cells. The level of endogenous TNF-α stimulated by jasminin was first analyzed by an enzyme-linked immunosorbent assay (ELISA) from the cell culture supernatant of RAW264.7 cells. The supernatants were then collected to investigate the potential antiviral effect against herpes simplex virus 1 (HSV-1). The antiviral effects of jasminin alone or its supernatants were evaluated by a plaque reduction assay. The potential activation of the PI3K-Akt pathway, three main mitogen-activated protein kinases (MAPKs), and nuclear factor (NF)-κB signaling pathways that induce TNF-α production were also investigated. Jasminin induces TNF-α protein expression in RAW264.7 cells without additional stimuli 10-fold more than the control. No significant up-expression of type I, II, and III interferons; interleukins 2 and 10; nor TNF-ß were observed by the jasminin stimuli. The supernatants, containing jasminin-induced-TNF-α, showed antiviral activity against HSV-1. The jasminin-stimulated cells caused the simultaneous activation of the Akt, MAPKs, and NF-κB signal pathways. Furthermore, the pretreatment of the cells with the Akt, MAPKs, and NF-κB inhibitors effectively suppressed jasminin-induced TNF-α production. Our research provides evidence that endogenous TNF-α can be used as a strategy to encounter viral infections. Additionally, the Akt, MAPKs, and NF-κB signaling pathways are involved in the TNF-α synthesis that induced by jasminin.

Drug Repositioning for Hand, Foot, and Mouth Disease.

Hand, foot, and mouth disease (HFMD) is a highly contagious disease in children caused by a group of enteroviruses. HFMD currently presents a major threat to infants and young children because of a lack of antiviral drugs in clinical practice. Drug repositioning is an attractive drug discovery strategy aimed at identifying and developing new drugs for diseases. Notably, repositioning of well-characterized therapeutics, including either approved or investigational drugs, is becoming a potential strategy to identify new treatments for virus infections. Various types of drugs, including antibacterial, cardiovascular, and anticancer agents, have been studied in relation to their therapeutic potential to treat HFMD. In this review, we summarize the major outbreaks of HFMD and the progress in drug repositioning to treat this disease. We also discuss the structural features and mode of action of these repositioned drugs and highlight the opportunities and challenges of drug repositioning for HFMD.

Immune Checkpoints in Viral Infections.

As evidence has mounted that virus-infected cells, such as cancer cells, negatively regulate the function of T-cells via immune checkpoints, it has become increasingly clear that viral infections similarly exploit immune checkpoints as an immune system escape mechanism. Although immune checkpoint therapy has been successfully used in cancer treatment, numerous studies have suggested that such therapy may also be highly relevant for treating viral infection, especially chronic viral infections. However, it has not yet been applied in this manner. Here, we reviewed recent findings regarding immune checkpoints in viral infections, including COVID-19, and discussed the role of immune checkpoints in different viral infections, as well as the potential for applying immune checkpoint blockades as antiviral therapy.

IKKε phosphorylates kindlin-2 to induce invadopodia formation and promote colorectal cancer metastasis.

Invadopodia formation is a key driver of cancer metastasis. The noncanonical IkB-related kinase IKKε has been implicated in cancer metastasis, but its roles in invadopodia formation and colorectal cancer (CRC) metastasis are unclear. Methods: Immunofluorescence, gelatin-degradation assay, wound healing assay and transwell invasion assay were used to determine the influence of IKKε over-expression, knockdown and pharmacological inhibition on invadopodia formation and the migratory and invasive capacity of CRC cells in vitro. Effects of IKKε knockdown or pharmacological inhibition on CRC metastasis were examined in mice. Immunohistochemistry staining was used to detect expression levels of IKKε in CRC patient tissues, and its association with prognosis in CRC patients was also analyzed. Immunoprecipitation, western blotting and in vitro kinase assay were constructed to investigate the molecular mechanisms. Results: IKKε co-localizes with F-actin and the invadopodia marker Tks5 at the gelatin-degrading sites of CRC cells. Genetic over-expression/knockdown or pharmacological inhibition of IKKε altered invadopodia formation and the migratory and invasive capacity of CRC cells in vitro. In vivo, knockdown or pharmacological inhibition of IKKε significantly suppressed metastasis of CRC cells in mice. IKKε knockdown also inhibited invadopodia formation in vivo. Clinical investigation of tumor specimens from 191 patients with CRC revealed that high IKKε expression correlates with metastasis and poor prognosis of CRC. Mechanistically, IKKε directly binds to and phosphorylates kindlin-2 at serine 159; this effect mediates the IKKε-induced invadopodia formation and promotion of CRC metastasis. Conclusions: We identify IKKε as a novel regulator of invadopodia formation and a unique mechanism by which IKKε promotes the metastasis of CRC. Our study suggests that IKKε is a potential target to suppress CRC metastasis.

Gypenoside L Inhibits Proliferation of Liver and Esophageal Cancer Cells by Inducing Senescence.

Senescence is an irreversible state of cell cycle arrest that can be triggered by multiple stimuli, such as oxygen reactive species and DNA damage. Growing evidence has proven that senescence is a tumor-suppressive approach in cancer treatment. Therefore, developing novel agents that modulate senescence may be an alternative strategy against cancer. In our study, we investigated the inhibitory effect of gypenoside L (Gyp-L), a saponin isolated from Gynostemma pentaphyllum, on cancer cell growth. We found that Gyp-L increased the SA-ß-galactosidase activity, promoted the production of senescence-associated secretory cytokines, and inhibited cell proliferation of human liver and esophageal cancer cells. Moreover, Gyp-L caused cell cycle arrest at S phase, and activated senescence-related cell cycle inhibitor proteins (p21 and p27) and their upstream regulators. In addition, Gyp-L activated p38 and ERK MAPK pathways and NF-κB pathway to induce senescence. Consistently, adding chemical inhibitors efficiently counteracted the Gyp-L-mediated senescence, growth inhibition, and cell cycle arrest in cancer cells. Furthermore, treatment with Gyp-L, enhanced the cytotoxicity of clinic therapeutic drugs, including 5-fluorouracil and cisplatin, on cancer cells. Overall, these results indicate that Gyp-L inhibits proliferation of cancer cells by inducing senescence and renders cancer cells more sensitive to chemotherapy.

Rho-kinase inhibitors from adlay seeds.

Rho-kinase enzymes are one of the most important targets recently identified in our bodies. Several lines of evidence indicate that these enzymes are involved in many diseases and cellular disorders. ROCK inhibitors may have clinical applications for cancer, hypertension, glaucoma, etc. Our study aims to identify the possible involvement of Rho-kinase inhibition to the multiple biological activities of adlay seeds and provide a rationale for their folkloric medicines. Hence, we evaluated Rho-kinase I and II inhibitory activity of the ethanol extract and 28 compounds derived from the seeds. A molecular docking assay was designed to estimate the binding affinity of the tested compounds with the target enzymes. The results of our study suggest a possible involvement of Rho-kinase inhibition to the multiple biological activities of the seeds. Furthermore, the results obtained with the tested compounds revealed some interesting skeletons as a scaffold for design and development of natural Rho-kinase inhibitors.

Anti-herpes simplex virus type 1 activity of Houttuynoid A, a flavonoid from Houttuynia cordata Thunb.

Early events in herpes simplex virus type 1 (HSV-1) infection reactivate latent human immunodeficiency virus, Epstein-Barr virus, and human papillomavirus in the presence of acyclovir (ACV). The common use of nucleoside analog medications, such as ACV and pencyclovir, has resulted in the emergence of drug-resistant HSV-1 strains in clinical therapy. Therefore, new antiherpetics that can inhibit early events in HSV-1 infection should be developed. An example of this treatment is Houttuynia cordata Thunb. water extract, which can inhibit HSV-1 infection through multiple mechanisms. In this study, the anti-HSV-1 activity of Houttuynoid A, a new type of flavonoid isolated from H. cordata, was investigated. Three different assays confirmed that this compound could exhibit strong in vitro anti-HSV-1 activity. One assay verified that this compound could inhibit HSV-1 multiplication and prevent lesion formation in a HSV-1 infection mouse model. Mechanism analysis revealed that this compound could inactivate HSV-1 infectivity by blocking viral membrane fusion. Moreover, Houttuynoid A exhibited antiviral activities against other alpha herpes viruses, such as HSV-2 and varicella zoster virus (VZV). In conclusion, Houttuynoid A may be a useful antiviral agent for HSV-1.

Fluorescence assay of dihydroorotate dehydrogenase that may become a cancer biomarker.

We developed an assay method for measuring dihydroorotate dehydrogenase (DHODH) activity in cultured HeLa cells and fibroblasts, and in stage III stomach cancer and adjacent normal tissues from the same patient. The assay comprised enzymatic reaction of DHODH with a large amount of dihydroorotic acid substrate, followed by fluorescence (FL) detection specific for orotic acid using the 4-trifluoromethyl-benzamidoxime fluorogenic reagent. The DHODH activities in the biologically complex samples were readily measured by the assay method. Our data indicate significantly higher DHODH activity in HeLa cells (340 ± 25.9 pmol/105 cells/h) than in normal fibroblasts (54.1 ± 7.40 pmol/105 cells/h), and in malignant tumour tissue (1.10 ± 0.19 nmol/mg total proteins/h) than in adjacent normal tissue (0.24 ± 0.11 nmol/mg total proteins/h). This is the first report that DHODH activity may be a diagnostic biomarker for cancer.

Tubulin polymerization-stimulating activity of Ganoderma triterpenoids.

Tubulin polymerization is an important target for anticancer therapies. Even though the potential of Ganoderma triterpenoids against various cancer targets had been well documented, studies on their tubulin polymerization-stimulating activity are scarce. This study was conducted to evaluate the effect of Ganoderma triterpenoids on tubulin polymerization. A total of twenty-four compounds were investigated using an in vitro tubulin polymerization assay. Results showed that most of the studied triterpenoids exhibited microtuble-stabilizing activity to different degrees. Among the investigated compounds, ganoderic acid T-Q, ganoderiol F, ganoderic acid S, ganodermanontriol and ganoderic acid TR were found to have the highest activities. A structure-activity relationship (SAR) analysis was performed. Extensive investigation of the SAR suggests the favorable structural features for the tubulin polymerization-stimulating activity of lanostane triterpenes. These findings would be helpful for further studies on the potential mechanisms of the anticancer activity of Ganoderma triterpenoids and give some indications on the design of tubulin-targeting anticancer agents.

Partial contribution of Rho-kinase inhibition to the bioactivity of Ganoderma lingzhi and its isolated compounds: insights on discovery of natural Rho-kinase inhibitors.

Recent studies identified Rho-kinase enzymes (ROCK-I and ROCK-II) as important targets that are involved in a variety of diseases. Synthetic Rho-kinase inhibitors have emerged as potential therapeutic agents to treat disorders such as hypertension, stroke, cancer, diabetes, glaucoma, etc. Our study is the first to screen the total ethanol extract of the medicinal mushroom Ganoderma lingzhi with thirty-five compounds for Rho-kinase inhibitory activity. Moreover, a molecular binding experiment was designed to investigate the binding affinity of the compounds at the active sites of Rho-kinase enzymes. The structure-activity relationship analysis was investigated. Our results suggest that the traditional uses of G. lingzhi might be in part due to the ROCK-I and ROCK-II inhibitory potential of this mushroom. Structure-activity relationship studies revealed some interesting features of the lanostane triterpenes that potentiate their Rho-kinase inhibition. These findings would be helpful for further studies on the design of Rho-kinase inhibitors from natural sources and open the door for contributions from other researchers for optimizing the development of natural Rho-kinase inhibitors.

Improved Biological Activities of Isoepoxypteryxin by Biotransformation.

Isoepoxypteryxin is the major coumarin of a Japanese medicinal plant Angelica shikokiana. This research was designed to study the effect of structural changes through fungal biotransformation on the reported biological activities of isoepoxypteryxin. Among the tested microorganisms, only Cordyceps sinensis had enzymes that could catalyze the ester hydrolysis and the reductive cleavage of the epoxide ring of isoepoxypteryxin, separately, to give two more polar metabolites (+)-cis-khellactone (P1) and a new coumarin derivative (+)-cis-3'-[(2-methyl-3-hydroxybutanoyl)oxy]-4'-acetoxy-3',4'-dihydroseselin (P2), respectively. The polar metabolite P2 showed stronger cytotoxicity and higher selectivity than isoepoxypteryxin. On the molecular level, P2 showed more in vitro inhibition of both tubulin polymerization and histone deacetylase 8 (HDAC8). Similarly, P2 showed more neuroprotection against amyloid beta fragment 1 - 42 (Aß1 - 42 )-induced neurotoxicity in human neuroblastoma cells (SH-SY5Y) and exhibited more inhibition of the in vitro aggregation of Aß1 - 42 . Both metabolites showed stronger antiplatelet aggregation by increased inhibition of thromboxane-A2 synthase (TXS) activity and thromboxane-A2 (TXA2) production. This study is the first to describe the improved cytotoxic, neuroprotective, and antiplatelet aggregation activities of isoepoxypteryxin through its biotransformation by C. sinensis.

Lucidumol C, a new cytotoxic lanostanoid triterpene from Ganoderma lingzhi against human cancer cells.

A new oxygenated lanostane-type triterpene, named lucidumol C, together with six known compounds, was isolated from the chloroform extract of the fruiting bodies of Ganoderma lingzhi. Structures were established based on extensive spectroscopic and chemical studies. Potential cytotoxic activities of the isolated compounds were evaluated against human colorectal carcinoma (HCT-116, Caco-2), human liver carcinoma (HepG2), and human cervical carcinoma (HeLa) cell lines using WST-1 reagent. Selectivity was evaluated using normal human fibroblast cells (TIG-1 and HF19). Among the compounds, lucidumol C showed potent selective cytotoxicity against HCT-116 cells with an IC50 value of 7.86 ± 4.56 µM and selectivity index (SI) >10 with remarkable cytotoxic activities against Caco-2, HepG2 and HeLa cell lines.

Biological Activities of Extracts from Different Parts of Cryptomeria japonica.

Cryptomeria japonica is one of the most important forest tree species in Japan. To increase the demand for domestic timber, broad uses related to the functional attributes of C. japonica must be developed. Several studies have examined the usefulness of C. japonica in terms of its biological activities, but a comprehensive study subjecting all parts of the C. japonica plant to the same solvent for extraction has not been done. Here, methanol extractions from the leaves, branches, bearing branches, male flowers, female flowers, cones, bark, heartwood, sapwood, pith, rhizomes, roots, and pollen were subjected to several in vitro assays of their biological activities such as antioxidant activity, anti-lipase activity, antibacterial activity and melanin-biosynthesis-inhibition activity. Their total phenolic content was also determined. The methanol extracts from each part of C. japonica except for pollen showed strong activities in the bioactivity assays. Furthermore, the methanol extracts were analyzed by GC/MS. The phytochemical profile varied among extracts from various parts of C. japonica. Our results suggest the great potential of C. japonica for use as a functional ingredient in health-related products.

DNA Aptamers in the Diagnosis and Treatment of Human Diseases.

Aptamers have a promising role in the field of life science and have been extensively researched for application as analytical tools, therapeutic agents and as vehicles for targeted drug delivery. Compared with RNA aptamers, DNA aptamers have inherent advantages in stability and facility of generation and synthesis. To better understand the specific potential of DNA aptamers, an overview of the progress in the generation and application of DNA aptamers in human disease diagnosis and therapy are presented in this review. Special attention is given to researches that are relatively close to practical application. DNA aptamers are expected to have great potential in the diagnosis and treatment of human diseases.

Multiple Biological Effects of Olive Oil By-products such as Leaves, Stems, Flowers, Olive Milled Waste, Fruit Pulp, and Seeds of the Olive Plant on Skin.

As olive oil production increases, so does the amount of olive oil by-products, which can cause environmental problems. Thus, new ways to utilize the by-products are needed. In the present study, five bioactive characteristics of olive oil by-products were assessed, namely their antioxidant, anti-bacterial, anti-melanogenesis, anti-allergic, and collagen-production-promoting activities. First, the extracts of leaves (May and October), stems (May and October), flowers, olive milled waste, fruit pulp and seeds were prepared using two safe solvents, ethanol and water. According to HPLC and LC/MS analysis and Folin-Ciocalteu assay, the ethanol extracts of the leaves (May and October), stems (May and October) and flowers contained oleuropein, and the ethanol extract of the stems showed the highest total phenol content. Oleuropein may contribute to the antioxidant and anti-melanogenesis activities of the leaves, stems, and flowers. However, other active compounds or synergistic effects present in the ethanol extracts are also likely to contribute to the anti-bacterial activity of the leaves and flowers, the anti-melanogenesis activity of some parts, the anti-allergic activity of olive milled waste, and the collagen-production-promoting activity of the leaves, stems, olive milled waste and fruit pulp. This study provides evidence that the by-products of olive oil have the potential to be further developed and used in the skin care industry.

Inhibition of enterovirus 71 replication by chrysosplenetin and penduletin.

In recent years, enterovirus 71 (EV71) infections have caused an increasing epidemic in young children, accompanying with more severe nervous system disease and more deaths. Unfortunately, there is no specific medication for it so far. Here we investigated the anti-EV71 activity of chrysosplenetin and penduletin, two o-methylated flavonols isolated from the leaves of Laggera pterodonta. These two compounds were found to have strong activity in vitro against EV71 with low cytotoxicity. In the cytopathic effect (CPE) inhibition assays, both plaque reduction assay and virus yield inhibition assay, the compounds showed a similar 50% inhibitory concentration (IC(50)) value of about 0.20 µM. The selectivity indices (SI) of chrysosplenetin and penduletin were 107.5 and 655.6 in African green monkey kidney (Vero) cells, and 69.5 and 200.5 in human rhabdomyosarcoma (RD) cells, accordingly. The preliminary mechanism analysis indicates that they function not through blocking virus entry or inactivating virus directly but inhibiting viral RNA replication. In the time-of-addition assay, both compounds inhibited progeny virus production and RNA replication by nearly 100% when introduced within 4h post infection. In addition to EV71, both compounds inhibited several other human enteroviruses with similar efficacy. These findings provide a significant lead for the discovery of anti-EV71 drug.

[Silencing HSV1 gD expression in cultured cells by RNA interference].

To explore the anti-HSV-1 effect of silencing gD gene expression by RNA interference, five 21-nucleotide duplex small interfering RNAs(siRNAs) targeting the HSV1 gD sequence were designed and the gD-EGFP fusion gene expression vector was constructed, then co-transfected into Vero cell, and screened the effective siRNA through analyzing the intensity of the EGFP fluorescence. Finally, the anti-HSV1 effect was confirmed by plaque reduction assay, real-time PCR and daughter virus titration of HSV1 infected Vero cells transfected with siRNAs. The study demonstrated that siRNAs could effectively and specifically inhibit gD gene expression in HSV1-infected cells, but only had a little effect on HSV1 infection, so taking gD as the target of siRNA against HSV1 needs further study.