Pharmacokinetics and antiangiogenic studies of potassium koetjapate in rats.

PURPOSE: Koetjapic acid is an active compound of a traditional medicinal plant, Sandoricum koetjape. Although koetjapic acid has a promising anticancer potential, yet it is highly insoluble in aqueous solutions. To increase aqueous solubility of koetjapic acid, we have previously reported a chemical modification of koetjapic acid to potassium koetjapate (KKA). However, pharmacokinetics of KKA has not been studied. In this study, pharmacokinetics and antiangiogenic efficacy of KKA are investigated. METHODS: Pharmacokinetics of KKA was studied after intravenous and oral administration in SD rats using HPLC. Anti-angiogenic efficacy of KKA was investigated in rat aorta, human endothelial cells (EA.hy926) and nude mice implanted with matrigel. RESULTS: Pharmacokinetic study revealed that KKA was readily absorbed into blood and stayed for a long time in the body with Tmax 2.89 ±â€¯0.12 h, Cmax 7.24 ±â€¯0.36 µg/mL and T1/2 1.46 ±â€¯0.03 h. The pharmacological results showed that KKA significantly suppressed sprouting of microvessels in rat aorta with IC50 18.4 ±â€¯4.2 µM and demonstrated remarkable inhibition of major endothelial functions such as migration, differentiation and VEGF expression in endothelial cells. Further, KKA significantly inhibited vascularization in matrigel plugs implanted in nude mice. CONCLUSIONS: The results indicate that bioabsorption of KKA from oral route was considerably efficient with longer retention in body than compared to that of the intravenous route. Further, improved antiangiogenic activity of KKA was recorded which could probably be due to its increased solubility and bioavailability. The results revealed that KKA inhibits angiogenesis by suppressing endothelial functions and expression of VEGF.

Proapoptotic and Antiangiogenic Activities of Arctium Lappa L. on Breast Cancer Cell Lines.

In this study, the bioactivity-guided fractionation was conducted on the aerial parts of Arctium lappa L. and then the extracts were tested in vitro on breast cancer (MCF-7), colorectal cancer (HCT-116), and normal cells (EA.hy926). The n-hexane fraction (EHX) of the ethanolic extract showed strong activity against both MCF-7 and EA.hy926 cell lines (IC50 values: 14.08 ± 3.64 and 27.25 ± 3.45 µg/mL, respectively). The proapoptotic activity of EHX was assessed using MCF-7. Morphological alterations were visualized using Hoechst staining and a transmission electron microscope. Cancer cell signal transduction pathways were investigated, and EHX significantly upregulated p53, TGF-ß, and NF-κB. Furthermore, EHX was found to disrupt the metastatic cascade of breast cancer cells by the inhibition of cell proliferation, migration, invasion, and colonization. The antiangiogenic activity of EHX fraction showed potent inhibition of rat aorta microvessels with IC50 value: 4.34 ± 1.64 µg/mL. This result was supported by the downregulation of VEGF-A expression up to 54%. Over 20 compounds were identified in EHX using GC-MS, of which stigmasterol, ß-sitosterol, and 3-O-acetyllupeol are the major active compounds. Phytochemical analysis of EHX showed higher phenolic and flavonoid contents with a substantial antioxidant activity. In conclusion, this work demonstrated that A. lappa has valuable anticancer activity and antiangiogenic properties that might be useful in breast cancer therapy.

Pleurotus sajor-caju can be used to synthesize silver nanoparticles with antifungal activity against Candida albicans.

BACKGROUND: Green synthesis of silver nanoparticles (AgNPs) has become widely practiced worldwide. In this study, AgNPs were synthesized using a hot-water extract of the edible mushroom Pleurotus sajor-caju. The product, PSC-AgNPs, was characterized by using UV-visible spectra, dynamic light scattering analysis, transmission electron microscopy (TEM), X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectrometry. To assess its antifungal activity against Candida albicans, gene transcription and protein expression analyses were conducted for CaICL1 and its product, ICL, using real-time quantitative polymerase chain reaction and western blot, respectively. RESULTS: PSC-AgNPs with an average particle size of 11.68 nm inhibited the growth of the pathogenic yeast C. albicans. Values for minimum inhibitory concentration and minimum fungicidal concentration were 250 and 500 mg L-1 , respectively. TEM images revealed that the average particle size of PSC-AgNPs was 16.8 nm, with the values for zeta potential and the polydispersity index being -8.54 mV and 0.137, respectively. XRD and FTIR spectra showed PSC-AgNPs to have a face-centered cubic crystalline structure. The polysaccharides and amino acid residues present in P. sajor-caju extract were found to be involved in reducing Ag+ to AgNP. Both CaICL1 transcription and ICL protein expression were found to be suppressed in the cells treated with PSC-AgNPs as compared with the control. CONCLUSION: Our PSC-AgNP preparation makes for a promising antifungal agent that can downregulate isocitrate lyase. © 2017 Society of Chemical Industry.

Effects of different mycotoxins on humans, cell genome and their involvement in cancer (Review).

The chemical nature of most of the mycotoxins makes them highly liposoluble compounds that can be absorbed from the site of exposure such as from the gastrointestinal and respiratory tract to the blood stream where it can be dissimilated throughout the body and reach different organs such as the liver and kidneys. Mycotoxins have a strong tendency and ability to penetrate the human and animal cells and reach the cellular genome where it causes a major mutagenic change in the nucleotide sequence which leads to strong and permanent defects in the genome. This defect will eventually be transcribed, translated and lead to the development of cancer. In this review, the chemical and physical nature of mycotoxins, the action of mycotoxins on the cellular genome and its effect on humans, mycotoxins and their carcinogenicity and mycotoxins research gaps are discussed, and new research areas are suggested. The research review posed various questions. What are the different mycotoxins that can cause cancer, what is the role of mycotoxins in causing cancer and what types of cancers can be caused by mycotoxins? These questions have been selected due to the significant increase in the mycotoxin contamination and the cancer incidence rate in the contemporary world. By revealing and understanding the role of mycotoxins in developing cancer, measures to reduce the risks and incidents of cancer could be taken.

Construction and Cloning of Reporter-Tagged Replicon cDNA for an In Vitro Replication Study of Murine Norovirus-1 (MNV-1).

BACKGROUND: A norovirus maintains its viability, infectivity and virulence by its ability to replicate. However, the biological mechanisms of the process remain to be explored. In this work, the NanoLuc™ Luciferase gene was used to develop a reporter-tagged replicon system to study norovirus replication. METHODS: The NanoLuc™ Luciferase reporter protein was engineered to be expressed as a fusion protein for MNV-1 minor capsid protein, VP2. The foot-and-mouth disease virus 2A (FMDV2A) sequence was inserted between the 3'end of the reporter gene and the VP2 start sequence to allow co-translational 'cleavage' of fusion proteins during intracellular transcript expression. Amplification of the fusion gene was performed using a series of standard and overlapping polymerase chain reactions. The resulting amplicon was then cloned into three readily available backbones of MNV-1 cDNA clones. RESULTS: Restriction enzyme analysis indicated that the NanoLucTM Luciferase gene was successfully inserted into the parental MNV-1 cDNA clone. The insertion was further confirmed by using DNA sequencing. CONCLUSION: NanoLuc™ Luciferase-tagged MNV-1 cDNA clones were successfully engineered. Such clones can be exploited to develop robust experimental assays for in vitro assessments of viral RNA replication.

Colorectal, Prostate and Pancreas Human Cancers Targeted Bioassay-guided Isolations and Characterization of Chemical Constituents from Tephrosia apollinea.

BACKGROUND: Cancer is characterized by uncontrolled cell division caused by dysregulation of cell proliferation. Therefore, agents that impair cancer cell proliferation could have potential therapeutic value. Higher plants are considered to be a good source of anticancer agents, and several clinically tested chemotherapeutic agents have been isolated from plants or derived from constituents of plant origin. METHODS: In the present study, a prenylated flavone (isoglabratephrin) was isolated from aerial parts of Tephrosia apollinea using a bioassay-guided technique. Chemical structure of the isolated compound was elucidated using spectroscopic techniques (NMR, IR, and LC-MC), elemental analysis and confirmed by using single crystal X-ray analysis. The antiproliferative effect of isoglabratephrin was tested using three human cancer cell lines (prostate (PC3), pancreatic (PANC-1), and colon (HCT-116) and one normal cell line (human fibroblast). RESULTS: Isoglabratephrin displayed selective inhibitory activity against proliferation of PC3 and PANC-1 cells with median inhibitory concentration values of 20.4 and 26.6 µg/ml, respectively. Isoglabratephrin demonstrated proapoptotic features, as it induced chromatin dissolution, nuclear condensation, and fragmentation. It also disrupted the mitochondrial membrane potential in the treated cancer cells. CONCLUSION: Isoglabratephrin could be a new lead to treat human prostate (PC3) and pancreatic (PANC-1) malignancies.

Cat's whiskers (Orthosiphon stamineus) tea modulates arthritis pathogenesis via the angiogenesis and inflammatory cascade.

BACKGROUND: Orthosiphon stamineus is used traditionally to treat gout, arthritis, and inflammatory related conditions. The in vitro anti-inflammatory effects of the plant have been scientifically investigated. The goal of the present study was to evaluate the potential of the 50% ethanol extract of O. stamineus (EOS) to treat rheumatoid arthritis. METHODS: Anti-arthritic activity was assessed using the in vitro heat denaturation test and the (FCA)-induced arthritis model. Efficacy was assessed by measurements of paw edema and granulation, X-ray radiography, fluorescence molecular tomography (FMT), and histological evaluation. Levels of (TNF-α), interleukin-1 (IL-1), and (COX-1 and COX-2) were analyzed in vitro in lipopolysaccharide (LPS)-stimulated human macrophage (U937). TNF-α and IL-1 levels in the serum samples of arthritic rats were also measured using an ELISA kit. RESULTS: Treatment with EOS resulted in dose-dependent inhibition of paw edema in acute and chronic models of inflammation. It also inhibited significantly the production of TNF-α, IL-1 COX-1, and COX-2 in the LPS-stimulated U937 macrophages. EOS significantly suppressed FCA-induced paw edema as well as the serum levels of TNF-α and IL-1. X-rays of the synovial joint of the hind leg showed considerable improvement in joint integrity and recovery of tibia-talus bones from degeneration and osteoporotic lesions. Histology of proximal interphalangeal joints of EOS-treated animals showed obvious protection of cartilage and soft tissue. Finally, FMT analysis strongly supported the anti-arthritic effect of EOS. EOS had high phenolic and total flavonoid content as well as strong antioxidant activity. CONCLUSIONS: Results illustrated that the anti-arthritic properties of O. stamineus could be beneficial for prevention and management of rheumatoid arthritis and other chronic inflammatory disorders. Illustration of the Anti- arthritis efficacy of Orthosiphon Stamineus standardized extract.

The Role of Isocitrate Lyase (ICL1) in the Metabolic Adaptation of Candida albicans Biofilms.

BACKGROUND: A major characteristic of Candida biofilm cells that differentiates them from free-floating cells is their high tolerance to antifungal drugs. This high resistance is attributed to particular biofilm properties, including the accumulation of extrapolymeric substances, morphogenetic switching, and metabolic flexibility. OBJECTIVES: This study evaluated the roles of metabolic processes (in particular the glyoxylate cycle) on biofilm formation, antifungal drug resistance, morphology, and cell wall components. METHODS: Growth, adhesion, biofilm formation, and cell wall carbohydrate composition were quantified for isogenic Candida albicans ICL1/ICL1, ICL1/icl1, and icl1/icl1 strains. The morphology and topography of these strains were compared by light microscopy and scanning electron microscopy. FKS1 (glucan synthase), ERG11 (14-α-demethylase), and CDR2 (efflux pump) mRNA levels were quantified using qRT-PCR. RESULTS: The ICL1/icl1 and icl1/icl1 strains formed similar biofilms and exhibited analogous drug-tolerance levels to the control ICL1/ICL1 strains. Furthermore, the drug sequestration ability of ß-1, 3-glucan, a major carbohydrate component of the extracellular matrix, was not impaired. However, the inactivation of ICL1 did impair morphogenesis. ICL1 deletion also had a considerable effect on the expression of the FKS1, ERG11, and CDR2 genes. FKS1 and ERG11 were upregulated in ICL1/icl1 and icl1/icl1 cells throughout the biofilm developmental stages, and CDR2 was upregulated at the early phase. However, their expression was downregulated compared to the control ICL1/ICL1 strain. CONCLUSIONS: We conclude that the glyoxylate cycle is not a specific determinant of biofilm drug resistance.

Scopoletin, an active principle of tree tobacco (Nicotiana glauca) inhibits human tumor vascularization in xenograft models and modulates ERK1, VEGF-A, and FGF-2 in computer model.

We recently reported the antineovascularization effect of scopoletin on rat aorta and identified its potential anti-angiogenic activity. Scopoletin could be useful as a systemic chemotherapeutic agent against angiogenesis-dependent malignancies if its antitumorigenic activity is investigated and scientifically proven using a suitable human tumor xenograft model. In the present study, bioassay-guided (anti-angiogenesis) phytochemical investigation was conducted on Nicotiana glauca extract which led to the isolation of scopoletin. Further, anti-angiogenic activity of scopoletin was characterized using ex vivo, in vivo and in silico angiogenesis models. Finally, the antitumorigenic efficacy of scopoletin was studied in human colorectal tumor xenograft model using athymic nude mice. For the first time, an in vivo anticancer activity of scopoletin was reported and characterized using xenograft models. Scopoletin caused significant suppression of sprouting of microvessels in rat aortic explants with IC50 (median inhibitory concentration) 0.06µM. Scopoletin (100 and 200mg/kg) strongly inhibited (59.72 and 89.4%, respectively) vascularization in matrigel plugs implanted in nude mice. In the tumor xenograft model, scopoletin showed remarkable inhibition on tumor growth (34.2 and 94.7% at 100 and 200mg/kg, respectively). Tumor histology revealed drastic reduction of the extent of vascularization. Further, immunostaining of CD31 and NG2 receptors in the histological sections confirmed the antivascular effect of scopoletin in tumor vasculature. In computer modeling, scopoletin showed strong ligand affinity and binding energies toward the following angiogenic factors: protein kinase (ERK1), vascular endothelial growth factor A (VEGF-A), and fibroblast growth factor 2 (FGF-2). These results suggest that the antitumor activity of scopoletin may be due to its strong anti-angiogenic effect, which may be mediated by its effective inhibition of ERK1, VEGF-A, and FGF-2.

Resistance of Candida albicans Biofilms to Drugs and the Host Immune System.

BACKGROUND: Candida albicans is a commensal fungus that resides on mucosal surfaces and in the gastrointestinal and genitourinary tracts in humans. However, it can cause an infection when the immune system of the host is impaired or if a niche becomes available. Many C. albicans infections are due to the organism's ability to form a biofilm on implanted medical devices. A biofilm represents an optimal medium for the growth of C. albicans as it allows cells to be enclosed by a self-produced extracellular matrix (ECM). OBJECTIVES: The present work investigated certain aspects of the resistance of C. albicans biofilms to drugs and the host immune system. RESULTS: An ECM was found to provide the infrastructure for biofilm formation, prevent disaggregation, and shield encapsulated C. albicans cells from antifungal drugs and the host's immune system. By influencing FKS1 and upregulating multiple glucan modification genes, ß-1, 3-glucan, an important component of ECM, was shown to be responsible for many of the biofilm's drug-resistant properties. On being engulfed by ECM, the fungal cell was found to switch from glycolysis to gluconeogenesis. Resembling the cellular response to starvation, this was followed by the activation of the glyoxylate cycle that allowed the use of simple molecules as energy sources. CONCLUSION: Mature biofilms were found to be much more resistant to antifungal agents and the host immune system than free cells. The factors responsible for high resistance included the complex architecture of biofilms, ECM, increased expression of drug efflux pumps, and metabolic plasticity.

Acute and Sub-Acute Toxicity Evaluation of the Methanolic Extract of Alstonia scholaris Stem Bark.

Alstonia scholaris has been used by traditional medicine practitioners since the medieval ages for the treatment of diseases. The aim of this research was to evaluate the acute and sub-acute oral toxicity of its methanolic extract. The acute toxicity test was conducted using Sprague Dawley (SD) rats. The methanolic extract of Alstonia scholaris stem bark (ASME) was administrated in a single dose of 2000 mg/kg via oral gavage; and the animals were observed for any behavioral changes or mortality. In the sub-acute toxicity study, SD rats received three doses of ASME (250, 500 and 1000 mg/kg) for 28 days via oral gavage. During these 28 days of treatment, the rats were observed weekly for toxicity symptoms. Following the 28-day treatment, the rats were sacrificed for hematological, biochemical and histopathology studies. In the acute toxicity study, Alstonia scholaris was found to be non-toxic at a dose of 2000 mg/kg b.w. In the sub-acute toxicity study, significant variations in body weight, hematological and biochemical parameters were observed in the experimental groups at the dose of 500 and 1000 mg/kg with the death of two female rats being recorded at the highest dose (1000 mg/kg b.w.). Histopathological studies revealed slight degeneration (lesion) and centrilobular necrosis in the liver, which was most expressed in the highest-dose group. These results demonstrate that, while a single dose and short term oral intake of Alstonia scholaris bark extract caused no toxicity up to a dose of 2000 mg/kg b.w., toxic effects manifested in the long term treatment at the highest dose (500 and 1000 mg/kg). The long-term toxic effect was found to be associated with alterations in hematological compositions and end-organ damage to the liver. Thus, prolonged use of high doses of ASME orally should be discouraged and lower doses encouraged.

The Anticancer, Antioxidant and Antimicrobial Properties of the Sesquiterpene ß-Caryophyllene from the Essential Oil of Aquilaria crassna.

The present study reports a bioassay-guided isolation of ß-caryophyllene from the essential oil of Aquilaria crassna. The structure of ß-caryophyllene was confirmed using FT-IR, NMR and MS. The antimicrobial effect of ß-caryophyllene was examined using human pathogenic bacterial and fungal strains. Its anti-oxidant properties were evaluated by DPPH and FRAP scavenging assays. The cytotoxicity of ß-caryophyllene was tested against seven human cancer cell lines. The corresponding selectivity index was determined by testing its cytotoxicity on normal cells. The effects of ß-caryophyllene were studied on a series of in vitro antitumor-promoting assays using colon cancer cells. Results showed that ß-caryophyllene demonstrated selective antibacterial activity against S. aureus (MIC 3 ± 1.0 µM) and more pronounced anti-fungal activity than kanamycin. ß-Caryophyllene also displayed strong antioxidant effects. Additionally, ß-caryophyllene exhibited selective anti-proliferative effects against colorectal cancer cells (IC50 19 µM). The results also showed that ß-caryophyllene induces apoptosis via nuclear condensation and fragmentation pathways including disruption of mitochondrial membrane potential. Further, ß-caryophyllene demonstrated potent inhibition against clonogenicity, migration, invasion and spheroid formation in colon cancer cells. These results prompt us to state that ß-caryophyllene is the active principle responsible for the selective anticancer and antimicrobial activities of A. crassnia. ß-Caryophyllene has great potential to be further developed as a promising chemotherapeutic agent against colorectal malignancies.