Targeting Wnt/tenascin C-mediated cross talk between pancreatic cancer cells and stellate cells via activation of the metastasis suppressor NDRG1.

A major barrier to successful pancreatic cancer (PC) treatment is the surrounding stroma, which secretes growth factors/cytokines that promote PC progression. Wnt and tenascin C (TnC) are key ligands secreted by stromal pancreatic stellate cells (PSCs) that then act on PC cells in a paracrine manner to activate the oncogenic ß-catenin and YAP/TAZ signaling pathways. Therefore, therapies targeting oncogenic Wnt/TnC cross talk between PC cells and PSCs constitute a promising new therapeutic approach for PC treatment. The metastasis suppressor N-myc downstream-regulated gene-1 (NDRG1) inhibits tumor progression and metastasis in numerous cancers, including PC. We demonstrate herein that targeting NDRG1 using the clinically trialed anticancer agent di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) inhibited Wnt/TnC-mediated interactions between PC cells and the surrounding PSCs. Mechanistically, NDRG1 and DpC markedly inhibit secretion of Wnt3a and TnC by PSCs, while also attenuating Wnt/ß-catenin and YAP/TAZ activation and downstream signaling in PC cells. This antioncogenic activity was mediated by direct inhibition of ß-catenin and YAP/TAZ nuclear localization and by increasing the Wnt inhibitor, DKK1. Expression of NDRG1 also inhibited transforming growth factor (TGF)-ß secretion by PC cells, a key mechanism by which PC cells activate PSCs. Using an in vivo orthotopic PC mouse model, we show DpC downregulated ß-catenin, TnC, and YAP/TAZ, while potently increasing NDRG1 expression in PC tumors. We conclude that NDRG1 and DpC inhibit Wnt/TnC-mediated interactions between PC cells and PSCs. These results further illuminate the antioncogenic mechanism of NDRG1 and the potential of targeting this metastasis suppressor to overcome the oncogenic effects of the PC-PSC interaction.

The metastasis suppressor NDRG1 directly regulates androgen receptor signaling in prostate cancer.

N-myc-downregulated gene 1 (NDRG1) has potent anticancer effects and inhibits cell growth, survival, metastasis, and angiogenesis. Previous studies suggested that NDRG1 is linked to the androgen signaling network, but this mechanistic relationship is unclear. Considering the crucial role of the androgen receptor (AR) in prostate cancer (PCa) progression, here we examined for the first time the effect of NDRG1 on AR expression, activation, and downstream signaling in LNCaP, 22Rv1, and C4-2B PCa cell types. We demonstrate that NDRG1 effectively promotes interaction of AR with the chaperone HSP90, which in turn stabilizes the AR while decreasing its androgen-mediated activation. The expression of NDRG1 suppressed: (1) AR activation, as measured by p-ARSer213 and p-ARSer81; (2) expression of a major AR transcriptional target, prostate-specific antigen (PSA); and (3) AR transcriptional activity, probably via inhibiting the c-Jun-AR interaction by reducing c-Jun phosphorylation (p-c-JunSer63). NDRG1 was also demonstrated to inhibit multiple key molecules involved in androgen-dependent and -independent signaling (namely EGFR, HER2, HER3, PI3K, STAT3, and NF-κB), which promote the development of castration-resistant prostate cancer. We also identified the cysteine-rich secretory protein/antigen 5/pathogenesis related-1 (CAP) domain of NDRG1 as vital for inhibition of AR activity. Examining NDRG1 and p-NDRG1 in PCa patient specimens revealed a significant negative correlation between NDRG1 and PSA levels in prostatectomy patients that went on to develop metastasis. These results highlight a vital role for NDRG1 in androgen signaling and its potential as a key therapeutic target and biomarker in PCa.

Breaking the cycle: Targeting of NDRG1 to inhibit bi-directional oncogenic cross-talk between pancreatic cancer and stroma.

Pancreatic cancer (PaCa) is characterized by dense stroma that hinders treatment efficacy, with pancreatic stellate cells (PSCs) being a major contributor to this stromal barrier and PaCa progression. Activated PSCs release hepatocyte growth factor (HGF) and insulin-like growth factor (IGF-1) that induce PaCa proliferation, metastasis and resistance to chemotherapy. We demonstrate for the first time that the metastasis suppressor, N-myc downstream regulated gene 1 (NDRG1), is a potent inhibitor of the PaCa-PSC cross-talk, leading to inhibition of HGF and IGF-1 signaling. NDRG1 also potently reduced the key driver of PaCa metastasis, namely GLI1, leading to reduced PSC-mediated cell migration. The novel clinically trialed anticancer agent, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), which upregulates NDRG1, potently de-sensitized PaCa cells to ligands secreted by activated PSCs. DpC and NDRG1 also inhibited the PaCa-mediated activation of PSCs via inhibition of sonic hedgehog (SHH) signaling. In vivo, DpC markedly reduced PaCa tumor growth and metastasis more avidly than the standard chemotherapy for this disease, gemcitabine. Uniquely, DpC was selectively cytotoxic against PaCa cells, while "re-programming" PSCs to an inactive state, decreasing collagen deposition and desmoplasia. Thus, targeting NDRG1 can effectively break the oncogenic cycle of PaCa-PSC bi-directional cross-talk to overcome PaCa desmoplasia and improve therapeutic outcomes.

Effect of Chronic Administration of Aqueous Leaves Extract of Moringa Stenopetala on Blood Parameters and Histology of Liver and Kidney in Rats.

BACKGROUND: Moringa stenopetala is used as nourishments, and treatment of various diseases. However, there is no much information on its safety. Hence, this study aimed to investigate the chronic administration of aqueous leaves extract of the plant. MATERIALS AND METHODS: Twenty-four rats were divided into: a control group administered with distilled water and three experimental groups, respectively, administered with the extract at doses of 500, 1000, and 2000 mg/kg orally for six months were investigated. Various hematological and biochemical parameters followed by histopathological analysis were evaluated. RESULTS: Treatment with the extract did not significantly affect most of the hematological parameters. However, there were a significant decrease of MCH at doses of 1000 mg/kg and 2000 mg/kg in male rats and increase of MCV at all doses in female rats. Levels of ALP at 2000 mg/kg and those of AST and ALT at 1000 and 2000 mg/kg were significantly increased in male rats. Furthermore, significant decrease in urea and increase in creatinine levels at the dose of 2000 mg/kg occurred in female rats. Mild histopathological changes were also observed in the liver of male rats and kidney of female rats treated with the extract, respectively at doses of 1000 and 2000 mg/kg, and 2000 mg/kg. CONCLUSION: Findings from the present study suggest that prolonged administration of extract of Moringa stenopetala at therapeutic doses is safe, but shows sign of mild toxicity as dose increases, with differential effect on male verses female rats.

Thiosemicarbazones suppress expression of the c-Met oncogene by mechanisms involving lysosomal degradation and intracellular shedding.

Considering the role of proto-oncogene c-Met (c-Met) in oncogenesis, we examined the effects of the metastasis suppressor, N-myc downstream-regulated gene-1 (NDRG1), and two NDRG1-inducing thiosemicarbazone-based agents, Dp44mT and DpC, on c-Met expression in DU145 and Huh7 cells. NDRG1 silencing without Dp44mT and DpC up-regulated c-Met expression, demonstrating that NDRG1 modulates c-Met levels. Dp44mT and DpC up-regulated NDRG1 by an iron-dependent mechanism and decreased c-Met levels, c-Met phosphorylation, and phosphorylation of its downstream effector, GRB2-associated binding protein 1 (GAB1). However, incubation with Dp44mT and DpC after NDRG1 silencing or silencing of the receptor tyrosine kinase inhibitor, mitogen-inducible gene 6 (MIG6), decreased c-Met and its phosphorylation, suggesting NDRG1- and MIG6-independent mechanism(s). Lysosomal inhibitors rescued the Dp44mT- and DpC-mediated c-Met down-regulation in DU145 cells. Confocal microscopy revealed that lysosomotropic agents and the thiosemicarbazones significantly increased co-localization between c-Met and lysosomal-associated membrane protein 2 (LAMP2). Moreover, generation of c-Met C-terminal fragment (CTF) and its intracellular domain (ICD) suggested metalloprotease-mediated cleavage. In fact, Dp44mT increased c-Met CTF while decreasing the ICD. Dp44mT and a γ-secretase inhibitor increased cellular c-Met CTF levels, suggesting that Dp44mT induces c-Met CTF levels by increasing metalloprotease activity. The broad metalloprotease inhibitors, EDTA and batimastat, partially prevented Dp44mT-mediated down-regulation of c-Met. In contrast, the ADAM inhibitor, TIMP metallopeptidase inhibitor 3 (TIMP-3), had no such effect, suggesting c-Met cleavage by another metalloprotease. Notably, Dp44mT did not induce extracellular c-Met shedding that could decrease c-Met levels. In summary, the thiosemicarbazones Dp44mT and DpC effectively inhibit oncogenic c-Met through lysosomal degradation and metalloprotease-mediated cleavage.

Acute and Subacute Toxicity of Methanol Extract of Syzygium guineense Leaves on the Histology of the Liver and Kidney and Biochemical Compositions of Blood in Rats.

Plant medicine is the oldest form of health care known to mankind. Syzygium guineense is one of the many species of Ethiopian medicinal plants which has a long history of use as remedies for various ailments such as dysentery, diarrhea, and hypertension. In many countries, herbal medicines and related products are introduced into the market without safety or toxicological evaluation. The aim of this study was to investigate the histopathological effect of 80% methanol extract of S. guineense on liver and kidney and blood parameters of rats. For acute toxicity study, rats were randomly divided into three groups (n=4). The control group received distilled water, while the experimental groups received a single dose of 2000 mg/kg and 5000 mg/kg 80% methanolic extract of S. guineense leaves per oral. For subacute toxicity study, the rats were randomly divided into three groups (n=6). The control group received distilled water, while the experimental groups received 500 mg/kg and 1500 mg/kg 80% methanol extract of S. guineense leaves orally for 28 days. At the end of the experiment, blood samples were collected for hematology and clinical chemistry evaluations. Gross pathology and histopathology of liver and kidneys were assessed. In the acute toxicity study, rats treated with 2000 mg/kg and 5000 mg/kg showed no toxicological signs observed on behavior, gross pathology, and body weight of rats. In the subacute toxicity study rats have showed no significant changes on behavior, gross pathology, body weight, and hematological and biochemical parameters, whereas both experimental groups had a lower blood glucose level compared with the control group (p < 0.05). There were no significant differences in the gross and histopathology of the liver and kidneys of experimental animals in extract exposed groups and their counterpart control. The 80% methanol extract of S. guineense does not produce adverse effects in rats after acute and subacute treatment. Before marketing a S. guineense leaf based remedy, subchronic and chronic toxicity evaluations need to be done.

The metastasis suppressor, NDRG1, attenuates oncogenic TGF-ß and NF-κB signaling to enhance membrane E-cadherin expression in pancreatic cancer cells.

The metastasis suppressor, N-myc downstream-regulated gene-1 (NDRG1), plays multifaceted roles in inhibiting oncogenic signaling and can suppress the epithelial mesenchymal transition (EMT), a key step in metastasis. In this investigation, NDRG1 inhibited the oncogenic effects of transforming growth factor-ß (TGF-ß) in PANC-1 pancreatic cancer cells, promoting expression and co-localization of E-cadherin and ß-catenin at the cell membrane. A similar effect of NDRG1 at supporting E-cadherin and ß-catenin co-localization at the cell membrane was also demonstrated for HT-29 colon and CFPAC-1 pancreatic cancer cells. The increase in E-cadherin in PANC-1 cells in response to NDRG1 was mediated by the reduction of three transcriptional repressors of E-cadherin, namely SNAIL, SLUG and ZEB1. To dissect the mechanisms how NDRG1 inhibits nuclear SNAIL, SLUG and ZEB1, we assessed involvement of the nuclear factor-κB (NF-κB) pathway, as its aberrant activation contributes to the EMT. Interestingly, NDRG1 comprehensively inhibited oncogenic NF-κB signaling at multiple sites in this pathway, suppressing NEMO, Iĸĸα and IĸBα expression, as well as reducing the activating phosphorylation of Iĸĸα/ß and IĸBα. NDRG1 also reduced the levels, nuclear co-localization and DNA-binding activity of NF-κB p65. Further, Iĸĸα, which integrates NF-κB and TGF-ß signaling to upregulate ZEB1, SNAIL and SLUG, was identified as an NDRG1 target. Considering this, therapies targeting NDRG1 could be a new strategy to inhibit metastasis, and as such, we examined novel anticancer agents, namely di-2-pyridylketone thiosemicarbazones, which upregulate NDRG1. These agents downregulated SNAIL, SLUG and ZEB1 in vitro and in vivo using a PANC-1 tumor xenograft model, demonstrating their marked potential.

Development, Characterization, and Evaluation of Novel Broad-Spectrum Antimicrobial Topical Formulations from Cymbopogon martini (Roxb.) W. Watson Essential Oil.

BACKGROUND: Skin infections were the most frequently encountered of all infections and the 4th leading cause of nonfatal disease burden. Topical drugs have been used for the management of skin infections. The growing concern of drug resistance to the topical agents has warned the need for continuous development of novel drug. Essential oils are the best candidate for new drug with different mode of action and target as they are rich in chemical constituents. OBJECTIVE: To evaluate and develop safe and effective topical antimicrobial formulations from essential oil of Cymbopogon martini. Method. Essential oil was extracted using hydrodistillation aerial part C. martini and topical formulations were prepared in five different semisolid bases. In vitro antimicrobial investigations were performed on essential oil and topical formulations. Skin sensitizations of the formulations were evaluated using guinea pig maximization. RESULTS: The essential oil of C. martini has shown broad-spectrum antimicrobial potency against all tested organisms with MIC value ranging from 0.65 to 10 µg/ml. Absolute inhibitions of growth of fungi were observed against Trichophyton mentagrophytes and Trichophyton rubrum at concentrations above 1% of oil and against Microsporum canis and Trichophyton verrucosum at a concentration of 4% oil. Among topical formulations, the highest antimicrobial activity was recorded in hydrophilic ointment followed by macrogol blend ointment. The antimicrobial activity of oil was higher in fungal pathogen compared to bacteria. Gram positive bacteria were more sensitive than gram negative bacteria. Hydrophilic and macrogol blend ointment containing 5% oil did not produce any skin sensitization on guinea pigs. CONCLUSION: In conclusion, topical formulations of C. martini essential oil can be alternative topical agents with safe broad-spectrum activity for the treatment of skin disorder. Further studies should focus on shelf life study and clinical study of the product.

Diuretic activity of the aqueous crude extract and hot tea infusion of Moringa stenopetala (Baker f.) Cufod. leaves in rats.

Moringa stenopetala (Baker f.) Cufod. is a medicinal plant that has been used for the treatment of different ailments such as hypertension and diabetes in Ethiopia. This study aims to assess the diuretic activity of the aqueous crude extract and hot tea infusion of M. stenopetala leaves in saline-loaded rats. Male Wistar rats were divided into ten groups (n = 5). The control group received distilled water (5 mL/kg), whereas the reference group received Furosemide (10 mg/kg). Groups III-X orally received different doses of aqueous crude extract (62.5, 125, 250, and 500 mg/kg) and hot tea infusion (1, 2, 4, and 6 teaspoons [Tsp]) based on community use. Urine volume was recorded every hour until the end of the 5th hour, and total urine volume of each animal was calculated. The diuretic activity and diuretic action were determined based on the urine output. Additionally, concentration of urinary sodium, chloride, and potassium ions was determined. The urinary Na+/K+ ratio and carbonyl anhydrase activity (Cl-/(Na+/K+)) were also assessed. The findings verified that the aqueous crude extract as well as the hot tea infusion of the leaves of M. stenopetala possesses significant (P < 0.01) diuretic, natriuretic, and kaliuretic effects. The aqueous crude extract (125 mg/kg) and hot tea infusion (2 Tsp) displayed the highest diuretic activity (101% and 96%, respectively) comparable to the reference drug, Furosemide (10 mg/kg). They also displayed a good natriuretic activity. The aqueous crude extract and hot tea infusion revealed a significant Na+ urinary excretion (P < 0.001) and Na+/K+ ratio (P < 0.05) at all test doses. There was also a significant (P < 0.01) Cl- urinary excretion at all test doses of aqueous crude extract except 62.5 mg/kg and all test doses of hot tea infusion except higher doses (4 and 6 Tsp). Thus, the aqueous crude extract as well as the hot tea infusion of the leaves of M. stenopetala causes a plausible increase in the urine volume and concentration of urinary electrolytes in rats.

Diuretic activity of the aqueous crude extract and solvent fractions of the leaves of Thymus serrulatus in mice.

The present study was undertaken to investigate the diuretic activity and acute toxicity profile of the crude aqueous extract and solvent fraction of the leaves of Thymus serrulatus in saline-loaded Swiss albino mice. Mice of either sex were divided into six groups (five animals in each group). The control group received normal saline (25 mL/kg), while the reference group received hydrochlorothiazide (10 mg/kg). Group III to Group VI received the test substances at dose levels of 125, 250, 500, and 1,000 mg/kg orally, respectively. At the end of the fifth hour, urine was collected, and total volume of urine excreted by each animal was recorded. Concentrations of urinary Na+ and K+ were determined, and the Na+/K+ ratio was calculated to make comparison among the groups. The acute toxicity of the most active fraction was also evaluated. The findings demonstrated that the crude aqueous extract of T. serrulatus leaves showed significant diuretic (P<0.01), natriuretic (P<0.01), and kaliuretic (P<0.01) effects. At the dose of 1,000 mg/kg, the n-butanol fraction demonstrated the highest diuretic activity comparable to the reference drug. It also showed a good natriuretic activity. The dichloromethane fraction, however, did not have significant diuretic activity. Both the crude aqueous extract of the leaves of T. serrulatus and its n-butanol fraction have diuretic activity with high concentration of urinary electrolytes in mice. Further studies, however, need to be pursued on the possible mechanism(s) of diuretic action.

Complementary Feeding: Review of Recommendations, Feeding Practices, and Adequacy of Homemade Complementary Food Preparations in Developing Countries - Lessons from Ethiopia.

Breastfeeding provides the ideal food during the first 6 months of life. Complementary feeding starts when breast milk is no longer sufficient by itself, where the target age is for 6-23 months. The gap between nutritional requirement and amount obtained from breast milk increases with age. For energy, 200, 300, and 550 kcal per day is expected to be covered by complementary foods at 6-8, 9-11, and 12-23 months, respectively. In addition, the complementary foods must provide relatively large proportions of micronutrients such as iron, zinc, phosphorus, magnesium, calcium, and vitamin B6. In several parts of the developing world, complementary feeding continues as a challenge to good nutrition in children. In Ethiopia, only 4.2% of breastfed children of 6-23 months of age have a minimum acceptable diet. The gaps are mostly attributed to either poor dietary quality or poor feeding practices, if not both. Commercial fortified foods are often beyond the reach of the poor. Thus, homemade complementary foods remain commonly used. Even when based on an improved recipe, however, unfortified plant-based complementary foods provide insufficient key micronutrients (especially, iron, zinc, and calcium) during the age of 6-23 months. Thus, this review assessed complementary feeding practice and recommendation and reviewed the level of adequacy of homemade complementary foods.

In vitro vasodilatory activity and possible mechanisms of the crude extracts and fractions of Moringa stenopetala (Baker f.) Cufod. leaves in isolated thoracic aorta of guinea pigs.

Moringa stenopetala, a plant belonging to the family of Moringaceae, is traditionally used for the treatment of hypertension and diabetes in Ethiopia. This study evaluates the in vitro vasodilatory effect of the extract of M. stenopetala leaves and the possible mechanisms in precontracted isolated thoracic aorta of guinea pigs. A guinea pig was sacrificed by gentle cervical dislocation, and the thoracic aortic ring was removed, cut spirally, and mounted in an organ bath containing Krebs-Henseleit physiological solution maintained at 37°C, and then the solution was aerated with carbogen (95% O2 and 5% CO2). The vasodilatory activity of cumulative doses of M. stenopetala extracts and fractions was evaluated on intact and denuded endothelium of isolated whole, spirally cut thoracic aortic strips of guinea pigs precontracted with potassium chloride (80 mM), epinephrine (1 µM), methylene blue (10 µM), and glibenclamide (10 µM) using polygraph. All extracts showed a relaxant effect in precontracted isolated whole, spirally cut thoracic aortic strips of guinea pigs in a dose-dependent manner, whereas the greater percentage of relaxant effect was shown with the addition of crude extracts in 80 mM of potassium chloride (99.10% and 95.56% for ethanol and aqueous crude extracts, respectively), and 1 µM of epinephrine (82.85% and 90.16% for ethanol and aqueous crude extracts, respectively) in precontracted isolated whole, spirally cut thoracic aortic strips of guinea pigs. Hence, the possible mechanism of relaxation might be mediated through the blockade of receptor-operated calcium influx and L-type voltage-dependent calcium channels. The aqueous extract showed more significant in vitro vasodilatory effect than its fractions and 70% ethanol extract.

Assessment of Caregiver's Knowledge, Complementary Feeding Practices, and Adequacy of Nutrient Intake from Homemade Foods for Children of 6-23 Months in Food Insecure Woredas of Wolayita Zone, Ethiopia.

Complementary feeding should fill the gap in energy and nutrients between estimated daily needs and amount obtained from breastfeeding from 6-month onward. However, homemade complementary foods are often reported for inadequacy in key nutrients despite reports of adequacy for energy and proteins. The aim of this study was to assess caregiver's complementary feeding knowledge, feeding practices, and to evaluate adequacy daily intakes from homemade complementary foods for children of 6-23 months in food insecure woredas of Wolayita zone, Ethiopia. A cross-sectional study assessing mothers/caregiver's knowledge and complementary feeding practice, adequacy of daily energy, and selected micronutrient intakes using weighed food record method. Multi-stage cluster sampling method was also used to select 68 households. Caregivers had good complementary feeding knowledge. Sixty (88.2%) children started complementary feeding at 6 months and 48 (70.6%) were fed three or more times per day. Daily energy intake, however, was significantly lower (p < 0.05) than estimated daily needs, with only 151.25, 253.77, and 364.76 (kcal/day) for 6-8, 9-11, and 12-23 months, respectively. Similarly, Ca and Zn intakes (milligrams per day) were below the daily requirements (p = 0.000), with value of 37.76, 0.96; 18.83, 1.21; 30.13, 1.96; for the 6-8, 9-11, and 12-23 months, respectively. Significant shortfall in daily intake of Fe (p = 0.000) was observed among the 6-8 and 9-11 months (3.25 and 4.17 mg/day, respectively), even accounting for high bioavailability. The complementary foods were energy dense. Daily energy, Ca, Zn, and Fe (except 12-23 months) intake, however, was lower than estimated daily requirements.

In vivo Antihypertensive and Antihyperlipidemic Effects of the Crude Extracts and Fractions of Moringa stenopetala (Baker f.) Cufod. Leaves in Rats.

BACKGROUND: Moringa stenopetala (Baker f.) Cufod. is a medicinal plant that has been used in Ethiopian traditional medicine as a remedy for treatment of hypertension and diabetes. The aim of this study was to evaluate antihypertensive and antihyperlipidemic effect in fructose induced hypertensive rats. METHODS: Rats were randomly divided into control and treatment groups (n = 6). Treatment groups were given daily extracts (250, 500, and 1000 mg/kg) orally with fructose. Whereas, positive, negative and normal control groups were received captopril (20 mg/kg/day with fructose), only fructose (66% w/v ad libitum) and distilled water ad libitum for 15 days, respectively. The blood pressure was measured every 5th day using tail cuff blood pressure analyzer, and on the 16th day the blood was sampled to evaluate antihyperlipidemic effect using clinical chemistry analyzer. RESULTS: The study showed that aqueous and 70% ethanol extracts significantly prevented blood pressure increment in a dose dependent manner comparable to that of the standard drug. Similarly, the extracts suppressed increment in lipid profile (cholesterol, glucose, and triglycerides) compared with negative control. The biochemical test revealed that extracts produced a rise in liver but no effect on kidney function indicators compared with normal control. CONCLUSION: These findings revealed that both crude extracts of M. stenopetala (Baker f.) Cufod. possess antihypertensive and antihyperlipidemic effect.