Repurposing synthetic and natural derivatives induces apoptosis in an orthotopic glioma-induced xenograft model by modulating WNT/ß-catenin signaling.

BACKGROUND: Glioblastomas arise from multistep tumorigenesis of the glial cells. Despite the current state-of-art treatment, tumor recurrence is inevitable. Among the innovations blooming up against glioblastoma, drug repurposing could provide profound premises for treatment enhancement. While considering this strategy, the efficacy of the repurposed drugs as monotherapies were not up to par; hence, the focus has now shifted to investigate the multidrug combinations. AIM: To investigate the efficacy of a quadruple-combinatorial treatment comprising temozolomide along with chloroquine, naringenin, and phloroglucinol in an orthotopic glioma-induced xenograft model. METHODS: Antiproliferative effect of the drugs was assessed by immunostaining. The expression profiles of WNT/ß-catenin and apoptotic markers were evaluated by qRT-PCR, immunoblotting, and ELISA. Patterns of mitochondrial depolarization was determined by flow cytometry. TUNEL assay was performed to affirm apoptosis induction. In vivo drug detection study was carried out by ESI-Q-TOF MS analysis. RESULTS: The quadruple-drug treatment had significantly hampered glioma proliferation and had induced apoptosis by modulating the WNT/ß-catenin signaling. Interestingly, the induction of apoptosis was associated with mitochondrial depolarization. The quadruple-drug cocktail had breached the blood-brain barrier and was detected in the brain tissue and plasma samples. CONCLUSION: The quadruple-drug combination served as a promising adjuvant therapy to combat glioblastoma lethality in vivo and can be probed for translation from bench to bedside.

A comparative clinical study on the generation of nitrosative stress in cataractous lenses of smokers and non-smoker tobacco patients.

AIM:: To quantify the levels of nitric oxide, inducible nitric oxide synthase, and 3-nitrotyrosine in cataractous lenses of smokers and smokers who chewed tobacco in comparison with non-smokers and non-smokers who chewed tobacco. STUDY DESIGN:: A total of 80 cataractous lenses from smokers, non-smokers, smokers with tobacco chewing habit, and non-smokers with tobacco chewing habit were collected from the patients who had enrolled in the Department of Ophthalmology, Mahatma Gandhi Medical College & Research Institute, Puducherry. METHODS:: Levels of nitric oxide, inducible nitric oxide synthase, and 3-nitrotyrosine were quantified using commercially available enzyme-linked immunosorbent assay kits. RESULTS:: The mean concentrations of lens nitric oxide, inducible nitric oxide synthase, and 3-nitrotyrosine are as follows: (a) smokers-112.01, 59.57, and 88.91 µmol/L; (b) smokers who chewed tobacco-175.15, 93.95, and 128.72 µmol/L; (c) non-smokers-76.15, 40.65, and 70.20 µmol/L; and (d) non-smokers who chewed tobacco-96.56, 52.87, and 83.88 µmol/L, respectively. CONCLUSION:: Nitric oxide, inducible nitric oxide synthase, and 3-nitrotyrosine at high levels are the major causative agents for cataractogenesis. The results of this study suggest that smoking and tobacco chewing habit generate nitrosative stress that could enhance the pathogenesis for early cataractogenesis.

Putative free radical-scavenging activity of an extract of Cineraria maritima in preventing selenite-induced cataractogenesis in Wistar rat pups.

PURPOSE: To investigate the possible free radical-scavenging activity of an extract of Cineraria maritima on selenite-induced cataractous lenses in Wistar rat pups. METHODS: In the present study, Wistar rat pups were divided into three experimental groups. On P10, Group I (control) rat pups received an intraperitoneal injection of 0.89% saline. Rats in groups II (selenite-challenged, untreated) and III (selenite-challenged, C. maritima treated) received a subcutaneous injection of sodium selenite (19 µmol/kg bodyweight); Group III rat pups also received an intraperitoneal injection of the extract of C. maritima (350 mg/kg bodyweight) once daily P9-14. Both eyes of each pup were examined from P16 until P30. Cytochemical localization of nitroblue tetrazolium salts and generation of superoxide, hydroxyl, and nitric oxide levels were measured. The expression of the inducible nitric oxide synthase gene was evaluated with reverse transcription-PCR. Immunoblot analysis was also performed to confirm the differential expression of the inducible nitric oxide synthase protein. RESULTS: Subcutaneous injection of sodium selenite led to severe oxidative damage in the lenticular tissues, shown by increased formation of formazan crystals, elevated generation of superoxide, hydroxyl, and nitric oxide radicals, and elevated inducible nitric oxide synthase gene and protein expression that possibly contributed to the opacification of the lens and thus cataract formation. When rat pups were treated with intraperitoneal administration of the extract of C. maritima, the generation of free radicals as well as the messenger ribonucleic acid and protein expression of inducible nitric oxide synthase were maintained at near normal levels. CONCLUSIONS: The data generated by this study suggest that an ethanolic extract of C. maritima possibly prevents cataractogenesis in a rat model by minimizing free radical generation.

Deciphering the potential efficacy of acetyl-L-carnitine (ALCAR) in maintaining connexin-mediated lenticular homeostasis.

PURPOSE: To determine the putative role of acetyl-L-carnitine (ALCAR) in maintaining normal intercellular communication in the lens through connexin. METHODS: In the present study, Wistar rat pups were divided into 3 groups of eight each. On postpartum day ten, Group I rat pups received an intraperitoneal injection (50 µl) of 0.89% saline. Rats in Groups II and III received a subcutaneous injection (50 µl) of sodium selenite (19 µmol/kg bodyweight); Group III rat pups also received an intraperitoneal injection of ALCAR (200 mg/kg bodyweight) once daily on postpartum days 9-14. Both eyes of each pup were examined from day 16 up to postpartum day 30. Alterations in the mean activity of the channel pumps, calcium-ATPase and sodium/potassium-ATPase, were determined. The expression of genes encoding key lenticular gap junctions (connexin 46 and connexin 50) and a channel pump (plasma membrane Ca(2+)-ATPase [PMCA1]) was evaluated by reverse transcription-PCR. Immunoblot analysis was also performed to confirm the differential expression of key lenticular connexin proteins. In addition, bioinformatics analysis was performed to determine the interacting residues of the connexin proteins with ALCAR. RESULTS: Significantly lower mean activities of Ca(2+)-ATPase and Na(+)/K(+) -ATPase were observed in the lenses of Group II rats than those in Group I rat lenses. However, the observed mean activities of Ca(2+)-ATPase and Na(+)/K(+)-ATPase in Group III rat lenses were significantly higher than those in Group II rat lenses. The mean mRNA transcript levels of the connexin 46 and connexin 50 genes were significantly lower, while the mean levels of PMCA1 gene transcripts were significantly higher, in Group II rat lenses than in Group I rat lenses. Immunoblot analysis also confirmed the altered expression of connexin proteins in lysates of whole lenses of Group II rats. However, the expression of connexin 46 and connexin 50 proteins in lenses from group III rats was essentially similar to that noted in lenses from normal (Group I) rats. Hydrogen bond-interaction between ALCAR and amino acid residues at the functional domain regions of connexin 46 and connexin 50 proteins was also demonstrated through bioinformatics tools. CONCLUSIONS: The results suggest that ALCAR plays a key role in maintaining lenticular homeostasis by promoting gap junctional intercellular communication.

Prevention of selenite-induced cataractogenesis by an ethanolic extract of Cineraria maritima: an experimental evaluation of the traditional eye medication.

In the present study, the antioxidant potential of an ethanolic extract of Cineraria maritima and its efficacy in preventing selenite-induced cataractogenesis were assessed in vitro and in vivo. In the in vitro phase of the study, lenses dissected out from the eyes of Wistar rats were incubated for 24 h at 37 °C in Dulbecco's modified Eagle medium (DMEM) alone (group I), in DMEM containing 100 µM of selenite only (group II), or in DMEM containing 100 µM of selenite and 300 µg/ml C. maritima extract added at the same time (group III). Gross morphological examination of the lenses revealed dense opacification in group II, minimal opacification in group III, and no opacification in group I lenses. The mean activities of the antioxidant enzymes catalase, glutathione peroxidase, and superoxide dismutase were significantly lower in group II than in group I or group III lenses, while malondialdehyde concentration was significantly higher in group II lenses than in group I and group III lenses. In the in vivo phase of the study, dense opacification of lenses was noted in all rat pups (100%) that had received a single subcutaneous injection of sodium selenite alone (19 µM/kg body weight) on postpartum day 10, whereas cataract formation occurred in only 33.3% of rat pups that had received selenite as well as an intraperitoneal injection of the extract of C. maritima (350 mg/kg body weight) for five consecutive days. These observations suggest that the ethanolic extract of C. maritima may prevent experimental selenite-induced cataractogenesis.