Functional characterization of a novel flavin reductase from a deep-sea sediment metagenomic library and its application for indirubin production.

Microbial synthesis is a desirable approach to produce indirubin but suffers from low synthetic efficiency. Insufficient supply of reduced flavins is one major factor limiting synthetic efficiency. To address this, a novel flavin reductase, MoxB, was discovered through screening of the metagenomic library. MoxB showed a strong preference for NADH over NADPH as the electron source for FMN/FAD reduction and exhibited the highest activity at pH 8.0 and 30°C. It displayed remarkable thermostability by maintaining 80% of full activity after incubation at 60°C for 1 h. Furthermore, MoxB showed great organic solvent tolerance and its activity could be significantly increased by bivalent metal ions. In addition, heterologous expression of the moxB gene in the indirubin-producing E. coli significantly improved indirubin production up to 15.12-fold. This discovery expands the understanding of flavin reductases and provides a promising catalytic tool for microbial indirubin production.IMPORTANCEMuch effort has been exerted to produce indirubin using engineered Escherichia coli, but high-level production has not been achieved so far. Insufficient supply of reduced flavins is one key factor limiting the catalytic efficiency. However, the flavin reductases involved in indirubin biosynthesis have not been hitherto reported. Discovery of the novel flavin reductase MoxB provides a useful tool for enhancing indirubin production by E. coli. Overexpression of MoxB in indirubin-producing E. coli increased indirubin production by 15.12-fold in comparison to the control strain. Our results document the function of flavin reductase that reduces flavins during indirubin biosynthesis and provide an important foundation for using the flavin reductases to improve indirubin production by engineered microorganisms.

Indirubin mediates adverse intestinal reactions in guinea pigs by downregulating the expression of AchE through AhR.

Indirubin is the main component of the traditional Chinese medicine Indigo naturalis (IN), a potent agonist of aryl hydrocarbon receptors (AhRs). In China, IN is used to treat psoriasis and ulcerative colitis, and indirubin is used for the treatment of chronic myelogenous leukaemia. However, IN and indirubin have adverse reactions, such as abdominal pain, diarrhoea, and intussusception, and their specific mechanism is unclear.The purpose of our research was to determine the specific mechanism underlying the adverse effects of IN and indirubin. By tracking the modifications in guinea pigs after the intragastric administration of indirubin for 28 days.The results demonstrate that indirubin could accelerate bowel movements and decrease intestinal acetylcholinesterase (AchE) expression. Experiments with NCM460 cells revealed that indirubin significantly reduced the expression of AchE, and the AchE levels were increased after the silencing of AhR and re-exposure to indirubin.This study showed that the inhibition of AchE expression by indirubin plays a key role in the occurrence of adverse reactions to indirubin and that the underlying mechanism is related to AhR-mediated AchE downregulation.

Indirubin regulates T cell differentiation by promoting αVß8 expression in bone marrow-derived dendritic cells to alleviate inflammatory bowel disease.

Inflammatory bowel disease is a disease that can invade the whole digestive tract and is accompanied by immune abnormalities. Immune dysfunction involving dendritic cells (DCs) and T cells is recognized as a key factor in diseases. Indirubin (IDRB) exerts antiinflammatory effects and can help in treating immune diseases. This study aimed to isolate bone marrow-derived dendritic cells (BMDCs) using lipopolysaccharide (LPS) to obtain mature DCs (mDCs). The expression of CD80, CD86, CD40, and MHC-II was detected using flow cytometry after treatment with IDRB. αVß8 siRNA was used to knock down αVß8 in mDCs, and the expression of CD80, CD86, CD40, and MHC-II was detected. Meanwhile, DCs were co-cultured with T cells. Then, T cell differentiation was detected using flow cytometry, and the cytokine levels were detected using enzyme-linked immunosorbent assay. The animal model of dextran sulfate sodium (DSS)-induced inflammatory bowel disease was established in mice. After intervention with IDRB and αVß8 shRNA, the intestinal tissues were evaluated using H&E staining, disease activity index (DAI) score, and histological damage index, and the corresponding factors and cytokines to regulatory T cells (Treg) and Th17 were measured. The results showed that αVß8 was expressed in immature DCs and mDCs. CD80, CD86, CD40, and MHC-II expression decreased after IDRB treatment in mDCs. Meanwhile, the expression of TNF-α and TGF-ß also decreased after IDRB treatment. The effect of IDRB on the expression of CD80, CD86, CD40, MHC-II, TNF-α, and TGF-ß in mDCs was reversed by αVß8 siRNA. The Treg differentiation increased after IDRB treatment, while the differentiation of Th17 cells was inhibited. This effect of IDRB was reversed by mDCs after treatment with αVß8 siRNA. In vivo experiments showed that IDRB alleviated the symptoms of inflammatory bowel disease in animals. Enteritis significantly reduced, and the effect of IDRB was reversed by αVß8 shRNA. The results suggested that IDRB regulated the differentiation of T cells by mediating the maturation of BMDCs through αVß8. This study confirmed the therapeutic effect of IDRB in inflammatory bowel disease and suggested that IDRB might serve as a potential drug.

Indirubin-3'-alkoxime derivatives for upregulation of Wnt signaling through dual inhibition of GSK-3ß and the CXXC5-Dvl interaction.

Glycogen synthase kinase-3ß (GSK-3ß) appears to be ordinarily expressed, and functionally redundant in Wnt/ß-catenin signaling. The Wnt proteins induce transduction of a cytoplasmic protein, Dishevelled (Dvl) which negatively modulates GSK-3ß activity. CXXC5 is a negative modulator of the Wnt/ß-catenin signaling through the interaction with Dvl in the cytosol. This indicates that Wnt/ß-catenin signaling could be efficiently modulated by controlling GSK-3ß and the CXXC5-Dvl interaction. In this study, we designed a series of indirubin-3'-oxime and indirubin-3'-alkoxime derivatives containing various functional groups at the 5- or 6-position (R1) alongside alkyl or benzylic moieties at the 3'-oxime position (R2). These activate Wnt signaling through inhibitions of both GSK-3ß and the CXXC5-Dvl protein-protein interaction, in addition, the improvement of pharmacological properties. The potent activity profiles of the synthesized compounds suggested that dual inhibition of GSK-3ß and the CXXC5-Dvl interaction could be an appropriate approach towards safely and efficientlyactivating Wntsignaling. Thus, dual-targeting inhibitors are potentially better candidates for efficient activation ofWntsignaling compared to GSK-3ß inhibitors.

Drug delivery of 6-bromoindirubin-3'-glycerol-oxime ether employing poly(D,L-lactide-co-glycolide)-based nanoencapsulation techniques with sustainable solvents.

BACKGROUND: Insufficient solubility and stability of bioactive small molecules as well as poor biocompatibility may cause low bioavailability and are common obstacles in drug development. One example of such problematic molecules is 6-bromoindirubin-3'-glycerol-oxime ether (6BIGOE), a hydrophobic indirubin derivative. 6BIGOE potently modulates the release of inflammatory cytokines and lipid mediators from isolated human monocytes through inhibition of glycogen synthase kinase-3 in a favorable fashion. However, 6BIGOE suffers from poor solubility and short half-lives in biological aqueous environment and exerts cytotoxic effects in various mammalian cells. In order to overcome the poor water solubility, instability and cytotoxicity of 6BIGOE, we applied encapsulation into poly(D,L-lactide-co-glycolide) (PLGA)-based nanoparticles by employing formulation methods using the sustainable solvents Cyrene™ or 400 g/mol poly(ethylene glycol) as suitable technology for efficient drug delivery of 6BIGOE. RESULTS: For all preparation techniques the physicochemical characterization of 6BIGOE-loaded nanoparticles revealed comparable crystallinity, sizes of about 230 nm with low polydispersity, negative zeta potentials around - 15 to - 25 mV, and biphasic release profiles over up to 24 h. Nanoparticles with improved cellular uptake and the ability to mask cytotoxic effects of 6BIGOE were obtained as shown in human monocytes over 48 h as well as in a shell-less hen's egg model. Intriguingly, encapsulation into these nanoparticles fully retains the anti-inflammatory properties of 6BIGOE, that is, favorable modulation of the release of inflammation-relevant cytokines and lipid mediators from human monocytes. CONCLUSIONS: Our formulation method of PLGA-based nanoparticles by applying sustainable, non-toxic solvents is a feasible nanotechnology that circumvents the poor bioavailability and biocompatibility of the cargo 6BIGOE. This technology yields favorable drug delivery systems for efficient interference with inflammatory processes, with improved pharmacotherapeutic potential.

Transdermal Delivery of Indirubin-Loaded Microemulsion Gel: Preparation, Characterization and Anti-Psoriatic Activity.

Psoriasis is an immune disease caused by rapid and incomplete differentiation of skin basal cells. Natural products such as indirubin have historically served as excellent sources for the treatments of psoriasis. However, the poor solubility and bioavailability due to its plane and rigid crystal structure, which limits its efficacy. Herein, to improve the efficacy of indirubin, a hydrogel-based microemulsion drug delivery system was developed for transdermal delivery. The mean droplet size of the optimized microemulsion was 84.37 nm, with a polydispersity index (PDI) less than 0.2 and zeta potential value of 0~-20 mV. The transdermal flux and skin retention of indirubin at 24 h were 47.34 ± 3.59 µg/cm2 and 8.77 ± 1.26 µg/cm2, respectively. The optimized microemulsion was dispersed in carbomer 934 hydrogel to increase the consistency. The indirubin-loaded microemulsion gel was tested on an imiquimod-induced psoriasis mouse model. Results showed that this preparation can improve psoriasis symptoms by down-regulating the expression of IL-17A, Ki67, and CD4+T. This experiment provides great scalability for researchers to treat psoriasis, avoid first-pass effects, and increase the concentration of targeted drugs.

Rosmarinus officinalis L. Leaf Extracts and Their Metabolites Inhibit the Aryl Hydrocarbon Receptor (AhR) Activation In Vitro and in Human Keratinocytes: Potential Impact on Inflammatory Skin Diseases and Skin Cancer.

Aryl hydrocarbon receptor (AhR) activation by environmental agents and microbial metabolites is potentially implicated in a series of skin diseases. Hence, it would be very important to identify natural compounds that could inhibit the AhR activation by ligands of microbial origin as 6-formylindolo[3,2-b]carbazole (FICZ), indirubin (IND) and pityriazepin (PZ) or the prototype ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Five different dry Rosmarinus officinalis L. extracts (ROEs) were assayed for their activities as antagonists of AhR ligand binding with guinea pig cytosol in the presence of [3H]TCDD. The methanolic ROE was further assayed towards CYP1A1 mRNA induction using RT-PCR in human keratinocytes against TCDD, FICZ, PZ, and IND. The isolated metabolites, carnosic acid, carnosol, 7-O-methyl-epi-rosmanol, 4',7-O-dimethylapigenin, and betulinic acid, were assayed for their agonist and antagonist activity in the presence and absence of TCDD using the gel retardation assay (GRA). All assayed ROE extracts showed similar dose-dependent activities with almost complete inhibition of AhR activation by TCDD at 100 ppm. The methanol ROE at 10 ppm showed 99%, 50%, 90%, and 85% inhibition against TCDD, FICZ, IND, and PZ, respectively, in human keratinocytes. Most assayed metabolites exhibited dose-dependent antagonist activity. ROEs inhibit AhR activation by TCDD and by the Malassezia metabolites FICZ, PZ, and IND. Hence, ROE could be useful for the prevention or treatment of skin diseases mediated by activation of AhR.

A Sensitive LC-MS/MS Method for the Simultaneous Determination of Two Thia-Analogous Indirubin N-Glycosides and Indirubin-3'-Monoxime in Plasma and Cell Culture Medium.

Indirubin was identified as an active component of Danggui Longhui Wan, an herbal mixture used in traditional Chinese medicine, and showed anticancer activity in clinical trials in patients with chronic leukemia. Investigations on the mechanisms of antitumor action of indirubins have mainly focused on the indirubin derivative indirubin-3'-monoxime (I3M). Meanwhile, antiproliferative and cytotoxic properties on cancer cells have also been demonstrated for several synthetic indirubin N-glycosides. In the present study, we demonstrate cytotoxic activity of the thia-analogous indirubin N-glycosides KD87 (3-[3'-oxo-benzo[b]thiophen-2'-(Z)-ylidene]-1-(ß-d-glucopyranosyl)-oxindole) and KD85 (3-[3'-oxo-benzo[b]thiophen-2'-(Z)-ylidene]-1-(ß-d-mannopyranosyl)-oxindole) against melanoma and squamous cell carcinoma cells as well as lung cancer and glioblastoma cells. The advanced state of preclinical studies on the effects of indirubins conducted to date underscores the need for pharmacokinetic data from cellular, animal, and human studies for which reliable quantification is required. Therefore, a sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the simultaneous measurement of KD87, KD85, and I3M in plasma and cell culture medium. Experimental conditions for sample preparation were optimized for human plasma protein precipitation and liquid-liquid extraction from plasma and cell culture medium. The methods were successfully validated in accordance with the U.S. Food and Drug Administration Bioanalytical Method Validation and evaluated for selectivity, sensitivity, matrix effect, recovery, carryover, calibration curve linearity, accuracy, precision, and stability. The applicability of the methods was demonstrated by the determination of KD87 in mouse plasma after prior intraperitoneal administration to mice.

[Optimization of extraction process of Children's Qingfei Zhisou Syrup based on pharmacodynamics].

This study determined the extraction rates of indirubin in Indigo Naturalis by ethanol reflux extraction method and water extraction method. The pharmacodynamic study against cough induced by ammonia water in the mouse model and the cough induced by citric acid in the guinea pig model were performed to optimize the extraction process of the sovereign medicinal Indigo Naturalis and the whole prescription of Children's Qingfei Zhisou Syrup. The extraction rate of indirubin by the ethanol reflux method was 51.89%, and indirubin was not detected in the product of water extraction. Two samples of Children's Qingfei Zhisou Syrup prepared with different methods can prolong the incubation period of cough and suppress the frequency of coughs in pharmacodynamic experiments. In terms of prolonging the incubation period of cough, the two samples prepared with different methods had no significant difference. In terms of reducing the frequency of coughs, the high-dose Five kinds of ethanol extracts such as indigo naturalis and three kinds of water extracts such as gypsum had better effect against the citric acid-induced cough of guinea pigs than other samples(P<0.05). The extraction rate of indirubin in Children's Qingfei Zhisou Syrup sample prepared with ethanol was higher than that with water. The two samples of Children's Qingfei Zhisou Syrup prepared with the two methods showed good antitussive effects. The sample prepared with 5 ingredients(including Indigo Naturalis) extracted with ethanol and 3 ingredients(including Gypsum Fibrosum) extracted with water had better alleviation effect on the citric acid-induced cough of guinea pig than the whole water extract sample. In conclusion, the optimum extraction scheme is ethanol extraction for 5 ingredients including Indigo Naturalis in combination with water extraction for 3 ingredients including Gypsum Fibrosum, and the Children's Qingfei Zhisou Syrup produced in this manner has better antitussive efficacy.

Indirubin-3'-monoxime induces paraptosis in MDA-MB-231 breast cancer cells by transmitting Ca2+ from endoplasmic reticulum to mitochondria.

PURPOSE: Indirubin-3'-monoxime (I3M) induces cell death in many cancer cells; however, whether I3M regulates paraptosis is unclear. The present study aimed to investigate I3M-induced paraptosis. METHODS: We treated various cancer cells with I3M, and measured vacuole formation (a paraptosis marker) and the regulating signaling pathway such as endoplasmic reticulum (ER) stress, reactive oxygen species, and proteasomal dysfunction. RESULTS: We found that I3M induced small vacuole formation in MDA-MB-231 breast cancer cells and transient knockdown of eIF2α and CHOP significantly downregulated vacuolation in the ER and mitochondria, as well as cell death in response to I3M, indicating that I3M-meditaed paraptosis was upregulated by ER stress. Moreover, I3M accumulated ubiquitinylated proteins via proteasome dysfunction, which stimulated ER stress-mediated Ca2+ release. A Ca2+ chelator significantly downregulated vacuolation in the ER and mitochondria as well as cell death, suggesting that Ca2+ was a key regulator in I3M-induced paraptosis. Our results also revealed that Ca2+ finally transited in mitochondria through mitochondrial Ca2+ uniporter (MCU), causing I3M-mediated paraptosis; however, the paraptosis was completely inhibited by, ruthenium red, an MCU inhibitor. CONCLUSION: I3M induced proteasomal dysfunction-mediated ER stress and subsequently promoted Ca2+ release, which was accumulated in the mitochondria via MCU, thus causing paraptosis in MDA-MB-231 breast cancer cells.

[Microbial community structure and transformation of indoles in soaking and fermentation of Indigo Naturalis].

The soaking and fermentation of Baphicacanthus cusia( Nees),the important intermediate link of Indigo Naturalis processing,facilitates the synthesis of indigo and indirubin precursors and the dissolution of endogenous enzymes and other effective components,while the role of microorganisms in the fermentation is ignored. The present study investigated the changes of microbial community structure in Indigo Naturalis processing based on 16 S amplicon sequencing and bioinformatics. Meanwhile,the contents of indigo,indirubin,isatin,tryptanthrin,indole glycoside,etc. were determined to explore the correlation between the microorganisms and the alterations of the main components. As demonstrated by the results,the microbial diversity decreased gradually with the fermentation,which bottomed out after the addition of lime. Proteobacteria,Bacteroidetes,and Firmicutes were the main dominant communities in the fermentation. The relative abundance of Proteobacteria declined gradually with the prolongation of fermentation time,and to the lowest level after the addition of lime. The relative abundance of Firmicutes increased,and that of Bacteroidetes decreased first and then increased. The contents of effective substances in Indigo Naturalis also showed different variation tendencies. As fermentation went on,indole glycoside decreased gradually; indigo first increased and then decreased; indirubin and isatin first decreased and then increased; tryptanthrin gradually increased. Those changes were presumedly related to the roles of microorganisms in the synthesis of different components. This study preliminarily clarified the important role of microorganisms in the soaking and fermentation and provided a scientific basis for the control of Indigo Naturalis processing and the preparation of high-quality Indigo Naturalis.

Inhibitory Effects of Indirubin-3'-oxime Derivatives on Lipid Accumulation in 3T3-L1 Cells.

Obesity elevates the risk of cardiovascular disease and has been strongly associated with increases in the incidence of many metabolic diseases. Therefore, prevention of obesity leads to the prevention of metabolic diseases. In light of this, substances that exert anti-obesity effects are crucial for the prevention of obesity. Indirubin, a 3,2' bisindole isomer of indigo, is the active component of the traditional Chinese medicine used for the treatment of chronic myelocytic leukemia. In particular, indirubin-3'-oxime (1) was shown to inhibit the differentiation of adipocytes. In this study, we investigated the inhibitory effects of nine indirubin-3'-oxime derivatives against lipid accumulation during differentiation in 3T3-L1 cells. Among the compounds tested, 5-methoxyindirubin-3'-oxime (2) and 6-bromoindirubin-3'-oxime (7) at 5 µM exhibited significantly stronger inhibitory activity than indirubin-3'-oxime (1). Furthermore, 5-methoxyindirubin-3'-oxime (2) and 6-bromoindirubin-3'-oxime (7) markedly suppressed the expression of CCAAT/enhancer-binding protein α, peroxisome proliferator activator γ2, and adipocyte protein 2, both of which are key adipogenic regulators at the intermediate stage of adipocyte differentiation. Our results demonstrate that 5-methoxyindirubin-3'-oxime (2) and 6-bromoindirubin-3'-oxime (7) significantly down-regulated lipid accumulation during differentiation of 3T3-L1 cells, suggesting their potential as novel therapeutic drugs against the development of obesity.

A novel small molecule A2A adenosine receptor agonist, indirubin-3'-monoxime, alleviates lipid-induced inflammation and insulin resistance in 3T3-L1 adipocytes.

Saturated free fatty acid-induced adipocyte inflammation plays a pivotal role in implementing insulin resistance and type 2 diabetes. Recent reports suggest A2A adenosine receptor (A2AAR) could be an attractive choice to counteract adipocyte inflammation and insulin resistance. Thus, an effective A2AAR agonist devoid of any toxicity is highly appealing. Here, we report that indirubin-3'-monoxime (I3M), a derivative of the bisindole alkaloid indirubin, efficiently binds and activates A2AAR which leads to the attenuation of lipid-induced adipocyte inflammation and insulin resistance. Using a combination of in silico virtual screening of potential anti-diabetic candidates and in vitro study on insulin-resistant model of 3T3-L1 adipocytes, we determined I3M through A2AAR activation markedly prevents lipid-induced impairment of the insulin signaling pathway in adipocytes without any toxic effects. While I3M restrains lipid-induced adipocyte inflammation by inhibiting NF-κB dependent pro-inflammatory cytokines expression, it also augments cAMP-mediated CREB activation and anti-inflammatory state in adipocytes. However, these attributes were compromised when cells were pretreated with the A2AAR antagonist, SCH 58261 or siRNA mediated knockdown of A2AAR. I3M, therefore, could be a valuable option to intervene adipocyte inflammation and thus showing promise for the management of insulin resistance and type 2 diabetes.

Development of UHPLC-MS/MS Method for Indirubin-3'-Oxime Derivative as a Novel FLT3 Inhibitor and Pharmacokinetic Study in Rats.

This study aimed to develop and validate a sensitive liquid chromatography-coupled tandem mass spectrometry method for the quantification of LDD-2614, an indirubin derivative and novel FLT3 inhibitor, in rat plasma. In addition, the developed analytical method was applied to observe the pharmacokinetic properties of LDD-2614. Chromatographic separation was achieved on a Luna omega C18 column using a mixture of water and acetonitrile, both containing 0.1% formic acid. Quantitation was performed using positive electrospray ionization in a multiple reaction monitoring (MRM) mode. The MRM transitions were optimized as m/z 426.2→113.1 for LDD-2614 and m/z 390.2→113.1 for LDD-2633 (internal standard), and the lower limit of quantification (LLOQ) for LDD-2614 was determined as 0.1 ng/mL. Including the LLOQ, the nine-point calibration curve was linear with a correlation coefficient greater than 0.9991. Inter- and intraday accuracies (RE) ranged from -3.19% to 8.72%, and the precision was within 9.02%. All validation results (accuracy, precision, matrix effect, recovery, stability, and dilution integrity) met the acceptance criteria of the U.S. Food and Drug Administration and the Korea Ministry of Food and Drug Safety guidelines. The proposed method was validated and demonstrated to be suitable for the quantification of LDD-2614 for pharmacokinetics studies.

Inhibitory effects of indirubin-3'-monoxime against human osteosarcoma.

Indirubin is widely used as the active component of "Dangui Luhui Wan" in ancient China. However, its effects against the osteosarcoma (OS), the most common primary malignancy, are still unknown. In our present study, we investigated the effects of the Indirubin-3'-monoxime (I3M), a derivative of indirubin with better water solubility, against the OS cells. We found I3M inhibited OS cell proliferation in a dose-dependent manner. Flow cytometry assays showed that I3M could not only induce OS cell apoptosis in a time- and dose-dependent manner but also regulate the cell cycle distribution. Additionally, we demonstrated that several Bcl-2 family members, cyclin-dependent kinases (CDKs) and cyclins contributed to this process. Furthermore, out data verified that I3M suppressed OS cell migration and invasion by decreasing MMP-2 and MMP-9 levels. Moreover, survivin and focal adhesion kinase (FAK) might play important roles in the anti-OS effects of I3M. The administration of I3M also inhibited the OS cell growth in mice. Taken together, our results indicated the inhibitory effects of I3M against human OS and thus might be an efficient candidate for OS chemotherapy.

Autoimmune thrombocytopenia: Current treatment options in adults with a focus on novel drugs.

Immune thrombocytopenia (ITP) is an autoimmune disease characterized by platelet destruction and reduced platelet production resulting in decreased platelet level and an increased risk of bleeding. Based on the immunologic mechanism of ITP, front-line standard therapy consists of corticosteroids and intravenous immunoglobulins (IVIG). If patients do not respond to the first-line treatment, or if continuous therapy is required, the disorder is called refractory ITP, and second-line therapy is indicated. This treatment may consist of rituximab, thrombopoietin receptor agonists, splenectomy, or cytotoxic drugs. Despite significant advances, many patients do not respond to any the treatments listed below, and new treatment options need to be developed for this relapsed and refractory group. Recent clinical studies have indicated promising outcomes for novel drugs, either as single agents or in combination with traditional drugs. This review discusses the latest and the most promising novel drugs for ITP in adults.

Preparation and investigation of indirubin-loaded SLN nanoparticles and their anti-cancer effects on human glioblastoma U87MG cells.

Indirubin, an ingredient in traditional Chinese medicine, is considered as an anti-cancer agent. However, due to its hydrophobic nature, clinical efficiency has been limited. Drug delivery via nanotechnology techniques open new windows toward treatment of cancerous patients. Glioblastoma multiforme (GBM) is the most severe and common type of brain primary tumors. Of common problems in targeting therapies of glioblastoma is the availability of drug in tumoric tissues. In this study, Indirubin loaded solid lipid nanoparticles were prepared and their therapeutic potentials and antitumoric effects were assessed on GBM cell line (U87MG). The SLNs were prepared with Cetyl palmitate and Polysorbat 80 via high-pressure homogenization (HPH) methods in hot mode. Then, properties of SLNs including size, zeta potential, drug encapsulation efficacy (EE %) and drug loading were characterized. SLNs morphology and size were observed using SEM and TEM. The crystalinity of formulation was determined by different scattering calorimetry (DSC). The amount of drug release and antitumor efficiency were evaluated at both normal brain pH of 7.2 and tumoric pH of 6.8. The prapared SLNs had mean size of 130 nm, zeta potential of -16 mV and EE of 99.73%. The results of DSC showed proper encapsulation of drug into SLNs. Drug release assessment in both pH displayed sustain release property. The result of MTT test exhibited a remarkable increment in antitumor activity of Indirubin loaded SLN in comparison with free form of drug and blank SLN on multiform GB. This study indicated that Indirubin loaded SLNs could act as a useful anticancer drugs.

A novel indirubin derivative that increases somatic cell plasticity and inhibits tumorigenicity.

Indirubin-based compounds affect diverse biological processes, such as inflammation and angiogenesis. In this study, we tested a novel indirubin derivative, LDD-1819 (2-((((2Z,3E)-5-hydroxy-5'-nitro-2'-oxo-[2,3'-biindolinylidene]-3-ylidene)amino)oxy)ethan-1-aminium chloride) for two major biological activities: cell plasticity and anti-cancer activity. Biological assays indicated that LDD-1819 induced somatic cell plasticity. LDD-1819 potentiated myoblast reprogramming into osteogenic cells and fibroblast reprogramming into adipogenic cells. Interestingly, in an assay of skeletal muscle dedifferentiation, LDD-1819 induced human muscle cellularization and blocked residual proliferative activity to produce a population of mononuclear refractory cells, which is also observed in the early stages of limb regeneration in urodele amphibians. In cancer cell lines, LDD-1819 treatment inhibited cell invasion and selectively induced apoptosis compared to normal cells. In an animal tumor xenograft model, LDD-1819 reduced human cancer cell metastasis in vivo at doses that did not produce toxicity. Biochemical assays showed that LDD-1819 possessed inhibitory activity against glycogen synthase kinase-3ß, which is linked to cell plasticity, and aurora kinase, which regulates carcinogenesis. These results indicate that novel indirubin derivative LDD-1819 is a dual inhibitor of glycogen synthase kinase-3ß and aurora A kinase, and has potential for development as an anti-cancer drug or as a reprogramming agent for cell-therapy based approaches to treat degenerative diseases.

Mechanism responsible for inhibitory effect of indirubin 3'-oxime on anticancer agent-induced YB-1 nuclear translocation in HepG2 human hepatocellular carcinoma cells.

YB-1 nuclear translocation/accumulation caused by anticancer agents leads to malignant transformation. Nuclear import of YB-1 requires a nuclear localization signal (YB-NLS). Previously, we identified five nucleocytoplasmic-shuttling proteins as YB-NLS binding proteins, and showed that they co-accumulate in the nucleus with YB-1 in response to treatment with actinomycin D. In addition, another group reported that transportin-1 is the molecule responsible for YB-1 nuclear translocation, binding to a region (PY-NLS) consistent with the YB-NLS. Recently, we found that indirubin 3'-oxime inhibits the nuclear localization of YB-1 in HepG2 cells and increases their sensitivity to actinomycin D. Here, we found that YB-1 nuclear translocation is dependent on the cellular mRNA level and that indirubin 3'-oxime inhibits the interaction between YB-1 and transportin-1. Interestingly, in cells showing inhibition of actinomycin D-induced YB-1 nuclear translocation by the compound, the YB-NLS-binding proteins as well as transportin-1 and its cargos were imported to the nucleus. Furthermore, the compound inhibited nuclear localization of the GFP-conjugated full-length YB-1 but not that of GFP-conjugated YB-NLS. These results indicate that indirubin 3'-oxime is a specific inhibitor of anticancer agent-induced YB-1 nuclear translocation, interacting with YB-1 itself in a region other than the YB-NLS/PY-NLS. This compound would increase the efficacy of cancer therapy.

Antifungal activity of selected Malassezia indolic compounds detected in culture.

BACKGROUND: Malassezia yeasts produce bioactive indolic substances when grown on L-tryptophan agar. A panel of these substances was tested against commensal and opportunistic fungi, the Minimum Inhibitory Concentration (MIC) was determined and the potential for in loco antifungal activity on the skin was assessed. MATERIALS AND METHODS: Eight indoles were included (malassezin, pityriacitrin, indirubin, indolo[3,2-b]carbazole, 6-formylindolo[3,2-b]carbazole, tryptanthrin, 6-hydroxymethylindolo[3,2-b]carbazole and 6-methylindolo[3,2-b]carbazole) and were tested against 40 fungal strains [yeasts: Malassezia spp.(N = 9); Cryptococcus spp.(N = 10); Candida spp.(N = 7); Yarrowia lipolytica(N = 1); Exophialla dermatitidis (N = 2); moulds: Aspergillus spp.(N = 7); Fusarium spp.(N = 2); Rhizopus oryzae(N = 2)]. The concentration of 5/8 of the tested indoles on diseased skin was calculated from published data. Kruskal-Wallis and Mann-Whitney U tests were employed for group susceptibility evaluation in 33 strains. RESULTS: The MIC range was 0.125-32 µg/mL, and the median log2 MIC was four. Indirubin was the most potent antifungal agent and differed significantly from the others. The highest median MIC was found for FICZ. Malassezia with Candida strains were more susceptible compared to Cryptococcus and Aspergillus, and this inhibitory activity was predicted to be valid also on human skin. CONCLUSIONS: Malassezia yeasts produce indolic species that inhibit an array of clinically significant yeasts and moulds.