Kaempferol and Biomodified Kaempferol from Sophora japonica Extract as Potential Sources of Anti-Cancer Polyphenolics against High Grade Glioma Cell Lines.

The enzymatic hydrolysis of the extract of Sophora japonica by two glycosyl hydrolases (hesperidinase and galactosidase) was performed in order to obtain kaempferol (KPF)-enriched extract with an enhanced anticancer activity. The current study examined the effectiveness of both Sophora japonica extracts (before (KPF-BBR) and after (KPF-ABR) bioconversion reactions) in reducing cell viability and inducing apoptosis in human high-degree gliomas in vitro. Cytotoxicity was determined using an MTT assay. The effects of both compounds on the proliferation of glioma cell lines were measured using trypan blue exclusion, flow cytometry for cell cycle, wound healing (WH), and neurosphere formation assays. Cellular apoptosis was detected by DNA fragmentation and phosphatidylserine exposure. qPCR and luciferase assays evaluated NF-kB pathway inhibition. The survival rate of NG-97 and U-251 cells significantly decreased in a time- and dose-dependent manner after the addition of KPF-BBR or KPF-ABR. Thus, a 50% reduction was observed in NG-97 cells at 800 µM (KPF-BBR) and 600 µM (KPF-ABR) after 72 h. Both compounds presented an IC50 of 1800 µM for U251 after 72 h. The above IC50 values were used in all of the following analyses. Neither of the KPF presented significant inhibitory effects on the non-tumoral cells (HDFa). However, after 24 h, both extracts (KPF-BBR and KPF-ABR) significantly inhibited the migration and proliferation of NG-97 and U-251 cells. In addition, MMP-9 was downregulated in glioma cells stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA) plus KPF-BBR and TPA+KPF-ABR compared with the TPA-treated cells. Both KPF-BBR and KPF-ABR significantly inhibited the proliferation of glioma stem cells (neurospheres) after 24 h. DNA fragmentation assays demonstrated that the apoptotic ratio of KPF-ABR-treated cell lines was significantly higher than in the control groups, especially NG-97, which is not TMZ resistant. In fact, the flow cytometric analysis indicated that KPF-BBR and KPF-ABR induced significant apoptosis in both glioma cells. In addition, both KPF induced S and G2/M cell cycle arrest in the U251 cells. The qPCR and luciferase assays showed that both KPFs downregulated TRAF6, IRAK2, IL-1ß, and TNF-α, indicating an inhibitory effect on the NF-kB pathway. Our findings suggest that both KPF-BBR and KPF-ABR can confer anti-tumoral effects on human cell glioma cells by inhibiting proliferation and inducing apoptosis, which is related to the NF-κB-mediated pathway. The KPF-enriched extract (KPF-ABR) showed an increased inhibitory effect on the cell migration and invasion, characterizing it as the best antitumor candidate.

Dipotassium Glycyrrhizininate Improves Skin Wound Healing by Modulating Inflammatory Process.

Wound healing is characterized by a systemic and complex process of cellular and molecular activities. Dipotassium Glycyrrhizinate (DPG), a side product derived from glycyrrhizic acid, has several biological effects, such as being antiallergic, antioxidant, antibacterial, antiviral, gastroprotective, antitumoral, and anti-inflammatory. This study aimed to evaluate the anti-inflammatory effect of topical DPG on the healing of cutaneous wounds by secondary intention in an in vivo experimental model. Twenty-four male Wistar rats were used in the experiment, and were randomly divided into six groups of four. Circular excisions were performed and topically treated for 14 days after wound induction. Macroscopic and histopathological analyses were performed. Gene expression was evaluated by real-time qPCR. Our results showed that treatment with DPG caused a decrease in the inflammatory exudate as well as an absence of active hyperemia. Increases in granulation tissue, tissue reepithelization, and total collagen were also observed. Furthermore, DPG treatment reduced the expression of pro-inflammatory cytokines (Tnf-α, Cox-2, Il-8, Irak-2, Nf-kB, and Il-1) while increasing the expression of Il-10, demonstrating anti-inflammatory effects across all three treatment periods. Based on our results, we conclude that DPG attenuates the inflammatory process by promoting skin wound healing through the modulation of distinct mechanisms and signaling pathways, including anti-inflammatory ones. This involves modulation of the expression of pro- and anti-inflammatory cytokine expression; promotion of new granulation tissue; angiogenesis; and tissue re-epithelialization, all of which contribute to tissue remodeling.

Evaluating Single-Nucleotide Variants in MicroRNA Targeting Sites and Mature MicroRNA In Vitro Cell Culture by Luciferase Reporter Gene Assays.

MicroRNAs (miRs) are small non-coding RNAs of 21-24 nucleotides in length that modulate gene expression by targeting the untranslated region (UTR) of mRNA. Single-nucleotide variants (SNVs) in primary miRs (pri-miRs), precursor miRs (pre-miRs), promoters of pri-miRs, and seed regions can affect miR stability or processing, may influence mature miR expression, and can affect target gene identification, respectively. The present protocol tests the binding and activity of miRs on 3'-UTR target sequences based on the expression of luciferase as a reporter gene fused to the UTR sequence in the presence of plasmids containing pre-miR of interest to test in vitro cell culture assay.

Anti-Migratory Effect of Dipotassium Glycyrrhizinate on Glioblastoma Cell Lines: Microarray Data for the Identification of Key MicroRNA Signatures.

The nuclear factor kappa B (NF-κB) pathway has been reported to be responsible for the aggressive disease phenomenon observed in glioblastoma (GBM). Dipotassium glycyrrhizinate (DPG), a dipotassium salt of glycyrrhizic acid isolated from licorice, has recently demonstrated an anti-tumoral effect on GBM cell lines U87MG and T98G through NF-κB suppression by IRAK2- and TRAF6-mediating microRNA (miR)-16 and miR-146a, respectively. Thus, the present study aimed to evaluate the expression profiles of miRNAs related to NF-κB suppression in T98G GBM cell line after DPG exposure using miRNA microarray (Affymetrix Human miRNA 4.0A), considering only predicted miRNAs as NF-κB regulator genes. Additional assays using U251 and U138MG cells were performed to validate the array results. DPG cytotoxicity was determined by (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, and cellular apoptosis was quantified by DNA fragmentation and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. The anti-proliferative effect was observed by cell proliferation and wound-healing assays, and the sphere formation assay examined whether DPG reduced stem cell subpopulation formation. The most over-expressed miRNAs were miR-4443 and miR-3620. The cytotoxic effect of DPG in U251 and U138MG was observed with an IC50 of 32 and 20 mM for 48 h, respectively. The IC50 of each cell line was used in all further assays. DPG treatment-induced apoptosis is observed by DNA fragmentation and increased TUNEL-positive cells. Cell proliferation and wound-healing assays showed an anti-proliferative and anti-migratory effect by DPG on the evaluated cell lines. In addition, DPG treatment led to a 100% reduction in sphere formation. The qPCR results in U251 and U138MG cells showed that DPG increased miR-4443 (2.44 vs. 1.11, p-value = 0.11; 8.27 vs. 1.25, p-value = 0.04) and miR-3620 expression (1.66 vs. 1.00, p-value = 0.03; 8.47 vs. 1.01, p-value = 0.03) and decreased CD209 (0.44 vs. 1.10, p-value = 0.03; 0.49 vs. 1.07, p-value = 0.04) and TNC (0.20 vs. 1.03, p-value = 0.001; 0.39 vs. 1.06, p-value = 0.01) mRNA levels compared to controls. Our results suggest that DPG inhibits cell viability by activating apoptosis and inhibiting cell proliferation and stem cell subpopulation formation through miR-4443 and miR-3620 upregulation. Both miRNAs are responsible for the post-transcriptional inhibition of NF-κB by CD209 and TNC modulation.

Dipotassium Glycyrrhizinate on Melanoma Cell Line: Inhibition of Cerebral Metastases Formation by Targeting NF-kB Genes-Mediating MicroRNA-4443 and MicroRNA-3620-Dipotassium Glycyrrhizinate Effect on Melanoma.

Glycyrrhizic acid (GA), a natural compound isolated from licorice (Glycyrrhiza glabra), has exhibited anti-inflammatory and anti-tumor effects in vitro. Dipotassium glycyrrhizinate (DPG), a dipotassium salt of GA, also has shown an anti-tumor effect on glioblastoma cell lines, U87MG and T98G. The study investigated the DPG effects in the melanoma cell line (SK-MEL-28). MTT assay demonstrated that the viability of the cells was significantly decreased in a time- and dose-dependent manner after DPG (IC50 = 36 mM; 24 h). DNA fragmentation suggested that DPG (IC50) induced cellular apoptosis, which was confirmed by a significant number of TUNEL-positive cells (p-value = 0.048) and by PARP-1 [0.55 vs. 1.02 arbitrary units (AUs), p-value = 0.001], BAX (1.91 vs. 1.05 AUs, p-value = 0.09), and BCL-2 (0.51 vs. 1.07 AUs, p-value = 0.0018) mRNA compared to control cells. The proliferation and wound-healing assays showed an anti-proliferative effect on DPG-IC50-treated cells, also indicating an inhibitory effect on cell migration (p-values < 0.001). Moreover, it was observed that DPG promoted a 100% reduction in melanospheres formation (p-value = 0.008). Our previous microRNAs (miRs) global analysis has revealed that DPG might increase miR-4443 and miR-3620 expression levels. Thus, qPCR showed that after DPG treatment, SK-MEL-28 cells presented significantly high miR-4443 (1.77 vs. 1.04 AUs, p-value = 0.02) and miR-3620 (2.30 vs. 1.00 AUs, p-value = 0.01) expression compared to control cells, which are predicted to target the NF-kB, CD209 and TNC genes, respectively. Both genes are responsible for cell attachment and migration, and qPCR revealed significantly decreased CD209 (1.01 vs. 0.54 AUs, p-value = 0.018) and TNC (1.00 vs. 0.31 AUs, p-value = 2.38 × 10−6) mRNA expression levels after DPG compared to untreated cells. Furthermore, the migration of SK-MEL-28 cells stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA) was attenuated by adding DPG by wound-healing assay (48 h: p-value = 0.004; 72 h: p-value = 7.0 × 10−4). In addition, the MMP-9 expression level was inhibited by DPG in melanoma cells stimulated by TPA and compared to TPA-treated cells (3.56 vs. 0.99 AUs, p-value = 0.0016) after 24 h of treatment. Our results suggested that DPG has an apoptotic, anti-proliferative, and anti-migratory effect on SK-MEL-28 cells. DPG was also able to inhibit cancer stem-like cells that may cause cerebral tumor formation.

The Anti-Inflammatory Properties of Licorice (Glycyrrhiza glabra)-Derived Compounds in Intestinal Disorders.

Intestinal diseases, such as inflammatory bowel diseases (IBDs) and colorectal cancer (CRC), are a significant source of morbidity and mortality worldwide. Epidemiological data have shown that IBD patients are at an increased risk for the development of CRC. IBD-associated cancer develops against a background of chronic inflammation and oxidative stress, and their products contribute to cancer development and progression. Therefore, the discovery of novel drugs for the treatment of intestinal diseases is urgently needed. Licorice (Glycyrrhiza glabra) has been largely used for thousands of years in traditional Chinese medicine. Licorice and its derived compounds possess antiallergic, antibacterial, antiviral, anti-inflammatory, and antitumor effects. These pharmacological properties aid in the treatment of inflammatory diseases. In this review, we discuss the pharmacological potential of bioactive compounds derived from Licorice and addresses their anti-inflammatory and antioxidant properties. We also discuss how the mechanisms of action in these compounds can influence their effectiveness and lead to therapeutic effects on intestinal disorders.

Natural Plant Compounds: Does Caffeine, Dipotassium Glycyrrhizinate, Curcumin, and Euphol Play Roles as Antitumoral Compounds in Glioblastoma Cell Lines?

Many plant-derived compounds are shown to be promising antitumor therapeutic agents by enhancing apoptosis-related pathways and cell cycle impairment in tumor cells, including glioblastoma (GBM) cell lines. We aimed to review four natural plant compounds effective in GBM cell lines as caffeine, dipotassium glycyrrhizinate (DPG), curcumin, and euphol. Furthermore, antitumoral effect of these plant compounds on GBM cell lines through microRNAs (miRs) modulation was investigated. However, only DPG and curcumin were found as effective on miR modulation. Caffeine arrests GBM cell cycle in G0/G1 phase by cyclin-dependent kinases (CDK) complex inhibition and by decreasing BCL-2 and increasing FOXO1 expression levels causing greater apoptotic activity. Caffeine can also directly inhibit IP3R3, p38 phosphorylation, and rho-associated protein kinase (ROCK), decreasing cell invasion and migration capacity or indirectly by inhibiting the tissue inhibitor metalloproteinase-1 (TIMP-1) and integrins ß1 and ß3, leading to lower matrix metalloproteinases, MMP-2 and MMP-9. DPG presents antitumoral effect in GBM cells related to nuclear factor kappa B (NF-κB) pathway suppression by IRAK2 and TRAF6-mediating miR-16 and miR-146a, respectively. More recently, it was observed that DPG upregulated miR-4443 and miR-3620, responsible for post-transcriptional inhibition of the NF-κB pathway by CD209 and TNC modulation, respectively leading to lower MMP-9 and migration capacity. Curcumin is able to increase miR-223-3p, miR-133a-3p, miR-181a-5p, miR-34a-5p, miR-30c-5p, and miR-1290 expression leading to serine or threonine kinase (AKT) pathway impairment and also it decreases miR-27a-5p, miR-221-3p, miR-21-5p, miR-125b-5p, and miR-151-3p expression causing p53-BCL2 pathway inhibition and consequently, cellular apoptosis. Interestingly, lower expression of miR-27a by curcumin action enhanced the C/EBP homologous protein(CHOP) expression, leading to paraptosis. Curcumin can inhibit miR-21 expression and consequently activate apoptosis through caspase 3 and death receptor (DR) 4 and 5 activation. Autophagy is controlled by the LC-3 protein that interacts with Atg family for the LC3-II formation and autophagy activation. Euphol can enhance LC3-II levels directly in GBM cells or inhibits tumor invasion and migration through PDK1 modulation.

The single nucleotide variant n.60G>C in the microRNA-146a associated with susceptibility to drug-resistant epilepsy.

OBJECTIVE: The aim of this study was to evaluate single nucleotide variants (SNVs) n.-411A > G (rs57095329) and n.60 G > C (rs2910164) in microRNA (miR)-146a, related to suppressing of TRAF6 with risk for epilepsy, as well as miR-146a and TRAF6 levels. METHODS: DNAs were extracted from epileptogenic tissues and blood leukocytes from drug-resistant epilepsy patients and healthy-individuals, respectively. Genotypes were identified by real-time PCR. Hardy-Weinberg equilibrium (HWE) and Fisher or X2 tests evaluated the difference between groups. The disease risk was assessed by odds ratio (OR) with 95 % confidence interval (95 %CI). The prognostic impact on probability seizure-free survival (PSF) was evaluated by Kaplan-Meier and log-rank tests. RESULTS: For rs57095329 both control and patient samples were not in HWE (p < 0.05) and the genotypes prevalence was similar in patients and controls (p>0.05). For rs2910164, control samples were in HWE (p = 0.61), contrasting with patients (p = 0.03), and similar frequencies of wild-type homozygous (GG) (43.4 % vs. 34.4 %, p = 0.2) and variant (CC) genotypes (8.0 % vs. 6.6 %, p = 0.6) were observed in patients and controls, respectively. However, increased frequency of heterozygous (GC) was observed in patients compared to controls (59.0 % vs. 42.7 %, p = 0.04) with 1.98 (95 %CI=1.09-3.57) risk for epilepsy. The miR-146a expression level in the epileptogenic tissues was lower in the GC (p = 0.02) and CC (p = 0.09) compared to GG genotype. TRAF6 expression level was higher in CC than in GG genotype (p = 0.09). Interestingly, there was an increased frequency of patients harboring GC genotype and less time until surgery compared to patients harboring GG or CC (36.06 % vs. 11.5 %, p = 0.01), confirmed by PSF (p = 0.04). CONCLUSIONS: The GC genotype for SNV rs2910164 appears associated with susceptibility to drug-resistant epilepsy due to the decreased MIR146a expression, favoring NF-kB pathway through TRAF6.

Growth Inhibitory Effects of Dipotassium Glycyrrhizinate in Glioblastoma Cell Lines by Targeting MicroRNAs Through the NF-κB Signaling Pathway.

It has been shown that nuclear factor kappa-B (NF-κB) is constitutively activated in glioblastoma (GBM), suggesting that the pathway could be a therapeutic target. Glycyrrhetic acid (GA), a compound isolated from licorice (Glycyrrhiza glabra), has been shown to decrease cell viability and increases apoptosis in human cancer cell lines by NF-κB signaling pathway suppression. Dipotassium glycyrrhizinate (DPG), a dipotassium salt of GA, has anti-inflammatory properties without toxicity. The current study examined the effectiveness of DPG as an anti-tumor in U87MG and T98G GBM cell lines. Additionally, we assessed DPG as a candidate for combinational therapy in GBM with temozolomide (TMZ). Our results demonstrated that the viability of U87MG and T98G cells significantly decreased in a time- and dose-dependent manner after DPG treatment, and the apoptotic ratio of DPG-treated groups was significantly higher than that of control groups. In addition, DPG in combination with TMZ revealed synergistic effects. Furthermore, the expression of NF-κB-luciferase-reporter in transfected GBM cell lines was remarkably reduced after DPG exposure by up-regulating miR16 and miR146a, which down-regulate its target genes, IRAK2 and TRAF6. A reduced neuro-sphere formation was also observed after DPG in both GBM cells. In conclusion, DPG presented anti-tumoral effect on GBM cell lines through a decrease on proliferation and an increase on apoptosis. In addition, our data also suggest that DPG anti-tumoral effect is related to NF-κB suppression, where IRAK2- and TRAF6-mediating miR16 and miR146a, respectively, might be a potential therapeutic target of DPG.

Optogenetics: the new molecular approach to control functions of neural cells in epilepsy, depression and tumors of the central nervous system.

The optogenetic tools have been described as valuable techniques to study neural activity through light stimulation, as well as potential neuromodulator approaches in the management of several central nervous system (CNS) diseases. Since the first bacteriorhodopsin protein described as a single-component light-activated regulator of transmembrane ion flow description, in 1980's, the focus has been on channel proteins for neurobiology; however, the advances in engineering techniques showed involvement changes in cellular biological behavior in several types of proteins involved in cell cytoskeleton regulation, motility and gene expression. Although the use of this technology has been published in many papers, a question still remains regarding real results and potential clinical applicability in CNS diseases, as well as the publications scarcity that systematically analyses the published results. Lastly, the aim of this review is to discuss the experimental results, molecular mechanisms and potential clinical applications of optogenetic tools in epilepsy and depression treatment, as well as its applicability in the treatment of CNS tumors.

Rare perianal extramammary Paget disease successfully treated using topical Imiquimod therapy.

BACKGROUND: Perianal Paget's disease (PPD) is a rare intraepithelial adenocarcinoma of the anal margin. Primary PPD likely represents intra-epithelial neoplasm from an apocrine source, whereas secondary disease may represent "pagetoid" spread from an anorectal malignancy. CASE PRESENTATION: Histologic CDX-2 and CK20 are hallmark markers for colorectal-derived Paget's cells. Interestingly, our primary PPD patient presented both positive and no internal malignancy was identified. In addition, a negative CK7 marker was observed in our case in contrast with previously reported. Surgical excision is the standard treatment; however, previous studies have demonstrated good response with Imiquimod 5% cream in patients with vulval extramammary Paget disease (EMPD). The efficiency of Imiquimod treatment for PPD has not been well described. Our PPD patient was successfully treated using Imiquimod 5% cream. CONCLUSIONS: This study describes a primary cutaneous PPD patient CDX-2+/CK20+/CK7- without invasion of the dermis and no associated colorectal carcinoma effectively treated using topical Imiquimod therapy, suggesting that Imiquimod might potentially be considered as a first-line treatment for PPD.