Discovering Potential Anti-Oral Squamous Cell Carcinoma Mechanisms from Kochiae Fructus Using Network-Based Pharmacology Analysis and Experimental Validation.

The natural product Kochiae Fructus (KF) is the ripe fruit of Kochia scoparia (L.) Schrad and is renowned for its anti-inflammatory, anticancer, anti-fungal, and anti-pruritic effects. This study examined the anticancer effect of components of KF to assess its potential as an adjuvant for cancer treatment. Network-based pharmacological and docking analyses of KF found associations with oral squamous cell carcinoma. The molecular docking of oleanolic acid (OA) with LC3 and SQSTM1 had high binding scores, and hydrogen binding with amino acids of the receptors suggests that OA is involved in autophagy, rather than the apoptosis pathway. For experimental validation, we exposed SCC-15 squamous carcinoma cells derived from a human tongue lesion to KF extract (KFE), OA, and cisplatin. The KFE caused SCC-15 cell death, and induced an accumulation of the autophagy marker proteins LC3 and p62/SQSTM1. The novelty of this study lies in the discovery that the change in autophagy protein levels can be related to the regulatory death of SCC-15 cells. These findings suggest that KF is a promising candidate for future studies to provide insight into the role of autophagy in cancer cells and advance our understanding of cancer prevention and treatment.

Bioengineering of Halobacterium sp. NRC-1 gas vesicle nanoparticles with GvpC fusion protein produced in E. coli.

Gas vesicle nanoparticles (GVNPs) are hollow, buoyant prokaryotic organelles used for cell flotation. GVNPs are encoded by a large gas vesicle protein (gvp) gene cluster in the haloarchaeon, Halobacterium sp. NRC-1, including one gene, gvpC, specifying a protein bound to the surface of the nanoparticles. Genetically engineered GVNPs in the Halobacterium sp. have been produced by fusion of foreign sequences to gvpC. To improve the versatility of the GVNP platform, we developed a method for displaying exogenously produced GvpC fusion proteins on the haloarchaeal nanoparticles. The streptococcal IgG-binding protein domain was fused at or near the C-terminus of GvpC, expressed and purified from E. coli, and shown to bind to wild-type GVNPs. The two fusion proteins, GvpC3GB and GvpC4GB, without or with a highly acidic GvpC C-terminal region, were found to be able to bind nanoparticles equally well. The GVNP-bound GvpC-IgG-binding fusion protein was also capable of binding to an enzyme-linked IgG-HRP complex which retained enzyme activity, demonstrating the hybrid system capability for display and delivery of protein complexes. This is the first report demonstrating functional binding of exogenously produced GvpC fusion proteins to wild-type haloarchaeal GVNPs which significantly expands the capability of the platform to produce bioengineered nanoparticles for biomedical applications. KEY POINTS: • Haloarchaeal gas vesicle nanoparticles (GVNPs) constitute a versatile display system. • GvpC-streptococcal IgG-binding fusion proteins expressed in E. coli bind to GVNPs. • IgG-binding proteins displayed on floating GVNPs bind and display IgG-HRP complex.

Inhibitory Effects of Porphyra tenera Extract on Oxidation and Inflammatory Responses.

Porphyra tenera (laver) has long been a popular and traditional seaweed food in Korea, Japan, and China. Historically, it was known as a marine medicinal herb to treat hemorrhoids and cholera morbus in Donguibogam. We investigated the effects of P. tenera extract (PTE) for its antioxidant and anti-inflammatory activities. These activities were measured using assays for 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitric oxide (NO) radical scavenging and its superoxide dismutase- (SOD-) like activity, and through the inhibitory production of inflammatory mediators (prostaglandin E2 (PGE2), NO, tumor necrosis factor alpha (TNF-α), and interleukin-6 (IL-6)) in lipopolysaccharide- (LPS-) stimulated Raw 264.7 cells. The antioxidant assay results showed that PTE displayed DPPH radical scavenging activity (46.44%), NO radical scavenging activity (67.14%), and SOD-like activity (80.29%) at a concentration of 5 mg/mL. In the anti-inflammatory assays, treatment with PTE (1 mg/mL) significantly inhibited expression levels of LPS-induced COX-2 and iNOS, as well as the production of PGE2, NO, TNF-α, and IL-6. These results show that PTE has antioxidant and anti-inflammatory properties and provide scientific evidence to explain the antioxidative and anti-inflammatory properties of PTE.

Astaxanthin Reduces Stemness Markers in BT20 and T47D Breast Cancer Stem Cells by Inhibiting Expression of Pontin and Mutant p53.

Astaxanthin (AST) is a product made from marine organisms that has been used as an anti-cancer supplement. It reduces pontin expression and induces apoptosis in SKBR3, a breast cancer cell line. Using Western blotting and qRT-PCR analyses, this study revealed that in the T47D and BT20 breast cancer cell lines, AST inhibits expression of pontin and mutp53, as well as the Oct4 and Nanog cancer stem cell (CSC) stemness genes. In addition, we explored the mechanism by which AST eradicates breast cancer cells using pontin siRNAs. Pontin knockdown by pontin siRNA reduced proliferation, Oct4 and Nanog expression, colony and spheroid formation, and migration and invasion abilities in breast cancer cells. In addition, reductions in Oct4, Nanog, and mutp53 expression following rottlerin treatment confirmed the role of pontin in these cells. Therefore, pontin may play a central role in the regulation of CSC properties and in cell proliferation following AST treatment. Taken together, these findings demonstrate that AST can repress CSC stemness genes in breast cancer cells, which implies that AST therapy could be used to improve the efficacy of other anti-cancer therapies against breast cancer cells.

Systemic Pharmacological Approach to Identification and Experimental Verification of the Effect of Anisi Stellati Fructus Extract on Chronic Myeloid Leukemia Cells.

Anisi stellati fructus (ASF) is the dried fruit of the Illicium verum Hook.f. tree. The aim of this research was to evaluate the antileukemic effect of ASF on chronic myeloid leukemia (CML) cells, which was hypothesized from the systemic pharmacological analysis of ASF, focusing on the combined effect of ASF extract (ASFE) and imatinib (IM). The compounds of ASF were identified using the Traditional Chinese Medicine Systems Pharmacology database and analysis platform. The target gene information was acquired from the UniProt database. The compound and target interaction network was generated from Cytoscape 3.7.1. Using this analysis, 10 compounds effective against CML cells were obtained. ASFE was prepared and analyzed by high-pressure liquid chromatography to provide experimental proof for the relationship between ASF and CML. The anti-p210Bcr-Abl effects of ASFE and ASFE + IM combination were evaluated by western blotting. Either ASFE alone or in combined treatment with IM on K-562 CML cells resulted in a significant reduction of the Bcr-Abl levels. As expected from the systemic analysis results, ASF had antileukemic activity, showing that it is a potential therapy for CML.

Amelioration of inflammatory responses by Socheongryong-Tang, a traditional herbal medicine, in RAW 264.7 cells and rats.

Socheongryong-Tang (SCRT) is a natural medicine prescription that has been mainly used in East Asia for the treatment of inflammatory disorders, including asthma and allergic rhinitis. The present study evaluated the anti-inflammatory effects of SCRT on lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and in a rat model of carrageenan (CA)-induced paw edema. Levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6 and prostaglandin E2 (PGE2) in the culture supernatant were quantified and nitric oxide (NO) production was monitored. In addition, the effect of SCRT on the protein expression of nuclear factor-κB (NF-κB), mitogen-activated protein kinases (MAPKs), inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) was assessed by western blot analysis. Furthermore, the effects of SCRT on acute inflammation in vivo and changes in the histomorphometry and histopathology of paw skin were observed using CA-treated rats. SCRT (1 mg/ml) inhibited the LPS-induced changes in the protein expression of NF-κB, JNK, ERK1/2, iNOS and COX-2, as well as the production of NO, PGE2 and cytokines. In the rat paw edema assay, administration of 1 g/kg of lyophilized powder obtained from the aqueous extracts of SCRT for 3 consecutive days inhibited the CA-induced increases in skin thickness, mast cell degranulation, and infiltration of inflammatory cells in the ventral and dorsal pedis skin within 4 h. These results demonstrated that SCRT exerts its anti-inflammatory activities in LPS-stimulated RAW 264.7 cells through decreasing the production of inflammatory mediators, including PGE2, NO and cytokines, via suppression of the NF-κB and JNK and ERK1/2 signaling pathways. In addition, the data of the CA-induced paw edema indicated an anti-edema effect of SCRT. SCRT (1 g/kg) reduced acute edematous inflammation through inhibition of mast cell degranulation and infiltration of inflammatory cells. Therefore, the present study provided scientific evidence for the anti-inflammatory activities of SCRT as well as the underlying mechanisms.

Biomolecular evidence of anti-inflammatory effects by Clematis mandshurica Ruprecht root extract in rodent cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Clematis mandshurica Ruprecht root is widely used in Asia as an analgesic and anti-inflammatory agent. This research investigated the anti-inflammatory effects of Clematis mandshurica Ruprecht root extract (CRE) using RAW 264.7 macrophage cells and carrageenan- (CA-) induced rat paw edema. MATERIALS AND METHODS: Production of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, nitric oxide (NO) and prostaglandin E2 (PGE2) in the culture supernatant, mRNA expression of TNF-α, IL-1ß, IL-6, iNOS and COX-2, protein expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) in the extract were assayed. In addition, the effect of CRE on acute inflammation in vivo was observed using CA-induced rat hind paw edema assay. The changes on the histopathology and histomorphometry of hind paw skins-dorsum and ventrum pedis were observed using CA-treated rats. RESULTS: Treatment with CRE (0.25, 0.5, and 1 mg/mL) resulted in inhibited levels of protein expression of lipopolysaccharide- (LPS-) induced iNOS, COX-2, NF-κB, and MAPKs (ERK, JNK, and p38) as well as production of TNF-α, IL-1ß, IL-6, NO, and PGE2 induced by LPS. Consistent with these results, CRE reduced the LPS-induced expressions of these cytokines, iNOS and COX-2 at the mRNA levels in a dose-dependent manner. In particular, results of the CA-induced rat hind paw edema assay showed an anti-edema effect of CRE. In addition, treatment with CRE resulted in dose-dependent inhibition of CA-induced increases of skin thickness, mast cell degranulation, and infiltrated inflammatory, TNF-α, IL-1ß, iNOS, and COX-2-positive cells in both dorsum and ventrum pedis skin, respectively. CONCLUSIONS: These results demonstrate that CRE exhibits anti-inflammatory activities via decreasing production of pro-inflammatory mediators through suppression of the pathways of NF-κB and MAPKs in LPS-induced macrophage cells. In addition, results of the CA-induced rat hind paw edema assay show an anti-edema effect of CRE. Our findings also support the traditional use of CRE in the inflammatory symptoms of rheumatic arthritis and acute icteric hepatitis. Thus, CRE may have therapeutic potential for a variety of inflammation-mediated diseases and may be developed into potent anti-inflammatory drugs.

Counteracting the activation of pAkt by inhibition of MEK/Erk inhibition reduces actin disruption-mediated apoptosis in PTEN-null PC3M prostate cancer cell lines.

The actin cytoskeleton is important in the maintenance of cellular homeostasis and in signal transduction pathways leading to cell growth and apoptotic cell death in eukaryotic cells. Disruption of actin dynamics is associated with morphological changes in cancer cells. Deletion of phosphatase and tensin homolog (PTEN), a tumor suppressor gene involved in the regulation of the cell cycle and apoptosis, leads to cytoskeleton disruption and double-strand breaks (DSBs). To study the mechanism(s) of actin disruption-mediated apoptosis and its potential application for anticancer therapy, PTEN-null PC3M prostate cancer cells were treated with latrunculin B (LB). LB induced destabilization of the actin microfilament and apoptosis in a dose-dependent manner, as demonstrated by morphological changes and nuclear condensation in the PC3M cells. In addition, it resulted in an increase in the levels of γH2AX recruitment, implicating the induction of DNA damage, including DSBs. Induction of Bax, with little effect on Bcl-2 expression, indicated that actin disruption causes apoptosis through activation of Bax signaling in PC3M cells. Treatment with U20126, a mitogen-activated protein kinase kinase (MEK) inhibitor, resulted in attenuated induction of DSBs and apoptosis through activation of protein kinase B (Akt), suggesting that LB-mediated actin dysfunction induces DSBs via the MEK/extracellular signal-regulated kinase (Erk) pathway in cells. Therefore, counteracting activation of phosphorylated Akt stemming from the inhibition of MEK/Erk resulted in attenuation of actin disruption-induced apoptotic events in the PC3M cells. The results of this study provide information not only for use in delineation of the molecular association between actin disruption and tumorigenesis, but also for the development of a strategy for actin-based anticancer chemotherapy against highly metastatic prostate cancer.