Exploitation of a novel phenothiazine derivative for its anti-cancer activities in malignant glioblastoma.

Glioblastoma remains the most malignant of all primary adult brain tumours with poor patient survival and limited treatment options. This study adopts a drug repurposing approach by investigating the anti-cancer activity of a derivative of the antipsychotic drug phenothiazine (DS00329) in malignant U251 and U87 glioblastoma cells. Results from MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and clonogenic assays showed that DS00329 inhibited short-term glioblastoma cell viability and long-term survival while sparing non-cancerous cells. Western blot analysis with an antibody to γH2AX showed that DS00329 induced DNA damage and flow cytometry and western blotting confirmed that it triggered a G1 cell cycle arrest which correlated with decreased levels in Cyclin A, Cyclin B, Cyclin D1 and cyclin dependent kinase 2 and an increase in levels of the cyclin dependent kinase inhibitor p21. DS00329 treated glioblastoma cells exhibited morphological and molecular markers typical of apoptotic cells such as membrane blebbing and cell shrinkage and an increase in levels of cleaved PARP. Flow cytometry with annexin V-FITC/propidium iodide staining confirmed that DS00329 induced apoptotic cell death in glioblastoma cells. We also show that DS00329 treatment of glioblastoma cells led to an increase in the autophagosome marker LC3-II and autophagy inhibition studies using bafilomycin A1 and wortmannin, showed that DS00329-induced-autophagy was a pro-death mechanism. Furthermore, DS00329 treatment of glioblastoma cells inhibited the phosphatidylinositol 3'-kinase/Akt cell survival pathway. Our findings suggest that DS00329 may be an effective treatment for glioblastoma and provide a rationale for further exploration and validation of the use of phenothiazines and their derivatives in the treatment of glioblastoma.

Corrigendum to "Antiproliferative and Apoptosis Induction Potential of the Methanolic Leaf Extract of Holarrhena floribunda (G. Don)".

[This corrects the article DOI: 10.1155/2015/756482.].

Cytotoxic and cell cycle arrest properties of two steroidal alkaloids isolated from Holarrhena floribunda (G. Don) T. Durand & Schinz leaves.

BACKGROUND: The plant Holarrhena floribunda (H. floribunda; G. Don) is indigenous to sub-Saharan Africa and is traditionally used to treat several ailments. The present study was carried out to isolate and characterize bioactive compounds with anti-proliferative activity present in H. floribunda extracts. METHODS: Compounds were isolated from H. floribunda using the bioassay-guided fractionation technique of repeated column chromatography and the step-wise application of the MTT reduction assay to assess antiproliferative bioactivity. The structures of the compounds were identified mainly using NMR. The effects of the isolated compounds on the viability, cell cycle and proliferation of human cancer cell lines (MCF-7, HeLa and HT-29) as well as the non-cancerous human fibroblast cell line (KMST-6) were investigated. RESULTS: Bioassay-guided fractionation yielded two steroidal alkaloids: holamine (1) and funtumine (2). The MTT reduction assay shows that both compounds exhibited selective dose-dependent cytotoxicity against the cancer cell lines studied. The isolated compounds induced cell cycle arrest at the G0/G1 and G2/M phases in the cancer cell lines with significant reduction in DNA synthesis. The results obtained show that the cancer cells (MCF-7, HeLa and HT-29) used in this study were more sensitive to the isolated compounds compared to the noncancerous fibroblast cells (KMST-6). CONCLUSION: The ability of the isolated compounds to cause cell cycle arrest and reduce DNA synthesis raises hopes for their possible development and use as potent anticancer drugs. However, more mechanistic studies need to be done for complete validation of the efficacy of the two compounds.

Antiproliferative and Apoptosis Induction Potential of the Methanolic Leaf Extract of Holarrhena floribunda (G. Don).

Natural plant products with potent growth inhibition and apoptosis induction properties are extensively being investigated for their cancer chemopreventive potential. Holarrhena floribunda (HF) is used in a wide range of traditional medicine practices. The present study investigated the antiproliferative and apoptosis induction potential of methanolic leaf extracts of HF against breast (MCF-7), colorectal (HT-29), and cervical (HeLa) cancer cells relative to normal KMST-6 fibroblasts. The MTT assay in conjunction with the trypan blue dye exclusion and clonogenic assays were used to determine the effects of the extracts on the cells. Caspase activities were assayed with Caspase-Glo 3/7 and Caspase-9 kits. Apoptosis induction was monitored by flow cytometry using the APOPercentage and Annexin V-FITC kits. Reactive oxygen species (ROS) was measured using the fluorogenic molecular probe 5-(and-6)-chloromethyl-2',7'-dichlorofluorescein diacetate acetyl ester and cell cycle arrest was detected with propidium iodide. Dose-response analyses of the extract showed greater sensitivity in cancer cell lines than in fibroblast controls. Induction of apoptosis, ROS, and cell cycle arrest were time- and dose-dependent for the cancer cell lines studied. These findings provide a basis for further studies on the isolation, characterization, and mechanistic evaluation of the bioactive compounds responsible for the antiproliferative activity of the plant extract.

Effect of quinine administration on Purkinje cells in the cerebellar cortex of adult Wistar rats.

OBJECTIVE: The effect of quinine commonly used for the treatment of Chloroquine resistant malaria and cerebral malaria on the population and transverse diameters of Purkinje cells in the cerebellar cortex was investigated. METHODS: Twenty-seven adult male wistar rats weighing between 150 g and 190 g were separated into three groups, each containing nine rats. The rats in group I were injected intramuscularly with equivalent volume of physiological saline, while group II rats were injected intramuscularly with an initial 20 mg/kg body weight dose of quinine followed by a 10 mg/kg body weight dose given 8 hourly for 7 days. The group III rats received the same treatment as group II, but were subjected to a withdrawal period of one week. The cerebellum of each rat was removed and fixed in 10% formol saline for routine histological procedures. RESULTS: The Purkinje cell population reduced significantly (P < 0.05) from the mean value of 363 +/- 5.2 cells/mm2 in group I to a mean value of 239 +/- 9.5 cells/mm2 in group II and 220 +/- 6.6 cells/mm2 in group III rats. The transverse diameters of the Purkinje cells also reduced significantly (p < 0.05) from the mean value of 1.20 +/- 0.02 microm in the group I to a mean value of 1.09 +/- 0.1 microm in group II and 0.75 +/- 0.03 microm in group III. CONCLUSION: The observed decrease in population and diameters of Purkinje cells in the treatment groups may impair cerebellar functions since they are the principal neurons of the cerebellum.

Comparative ultrastructural analyses of platelets and fibrin networks using the murine model of asthma.

The murine Balb/c asthma model has been used successfully for a number of in vivo immunological applications and for testing novel therapeutics, and it is a reliable, clinically relevant facsimile of the human disease. Here we investigate whether this model can be used to study other components of the human body, e.g. ultrastructure. In particular, we investigate the effect of the phytomedicine Euphorbia hirta (used to treat asthma), on the ultrastructure of fibrin as well as platelets, cellular structures that both play an important role in the coagulation process. Hydrocortisone is used as positive control. Ultrastructure of the fibrin networks and platelets of control mice were compared to mice that were asthmatic, treated with two concentrations of hydrocortisone and one concentration of the plant material. Results indicate control mice possess major, thick fibers and minor thin fibers as well as tight round platelet aggregates with typical pseudopodia formation. Minor fibers of asthmatic mice have a netlike appearance covering the major fibers, while the platelets seem to form loosely connected, granular aggregates. Both concentrations of hydrocortisone make the fibrin more fragile and that platelet morphology changes form a tight platelet aggregate to a more granular aggregate not closely fused to each other. We conclude that E. hirta does not impact on the fragility of the fibrin and that it prevents the minor fibers to form the dense netlike layer over the major fibers, as is seen in untreated asthmatic mice. This ultrastructural morphology might give us better insight into asthma and the possible new treatment regimes.

Comparative ultrastructural analyses of mouse, rabbit, and human platelets and fibrin networks.

Platelets and fibrin play an important role in the coagulation process, where they are involved in the maintenance of hemostasis. Fibrin dysfunction is associated with the development of vascular complications, while proneness to the formation of tight and rigid fibrin networks is independently associated with thrombotic disease. Here we investigate the ultrastructure of human, rabbit, and mouse platelets and fibrin networks, using the scanning electron microscope. Human and rabbit fibrin and platelets, with regards to morphology as well as size of major and minor fibers compare well with each other. However, mouse fibers are much thinner and form a flimsy branching network. Platelet aggregate morphology of all three species compare well with each other. We conclude that rabbit platelet and fibrin networks could be used successfully when studying the effect of pharmaceutical products in preclinical trials, when looking at the effects of these products on morphology and ultrastructure.

Curcuma longa extract as a histological dye for collagen fibres and red blood cells.

Crude ethanolic extract and column chromatographic fractions of the Allepey cultivar of Curcuma longa Roxb, commonly called turmeric (tumeric) in commerce, were used as a stain for tissue sections. Staining was carried out under basic, acidic and neutral media conditions. Inorganic and organic dissolution solvents were used. The stain was used as a counterstain after alum and iron haematoxylins. C. longa stained collagen fibres, cytoplasm, red blood cells and muscle cells yellow. It also stained in a fashion similar to eosin, except for its intense yellow colour. Preliminary phytochemical evaluation of the active column fraction revealed that it contained flavonoids, free anthraquinone and deoxy sugar. A cheap, natural dye can thus be obtained from C. longa.

Neuroprotective effect of dexamethasone on the morphology of the Irradiated post natal developing cerebellum of wistar rat (Rattus norvegicus)

RESUMEN: Se estudió el efecto neuroprotector de la dexametasona, sobre el cerebelo post-natal en desarrollo irradiado de ratas Wistar. 75 neonatos de 1 día de edad fueron separados en 3 grupos; el grupo control no recibió ni drogas ni irradiación, un grupo irradiado y el otro irradiado con aplicación de dexametasona. Esta droga fue administrada una hora antes de la exposición de 5Gray (5Gy) de rayos gamma. El tejido cerebelar de cada grupo con 5, 9, 14, 21 y 25 días fueron procesados para estudios histológicos e histomorfométricos. El resultado del estudio demostró que la sola irradiación redujo significativamente el grosor de la capa granular externa, en los grupos con 5 y 14 día,s con un p0,05; la capa molecular en los ejemplares de 5, 9, 14 y 21 días con un p0,05 y la capa granular en las ratas de 5,9,14 y 25 días, con un p0,05. Cuando se combinó la dexametasona con irradiación, se observó un grosor significativamente diferente en la capa granular externa, en especímenes con 5, 9 y 14 días; en la capa molecular en los animales de 5, 14 y 21 días y en la capa granular en los que tenían 5 y 14 días, al compararlos con el grupo irradiado, con un p>0,05. El diámetro de las células de Purkinje (capa de Purkinje) aunque fue significativamente reducido en el grupo irradiado de 14 y 21 días, no fue significativamente diferente cuandos se administró dexametasona a los animales irradiados de 5, 9, 14, 21 y 25 días con un p0,05. Histológicamente, las células de la capa molecular, en el grupo irradiado de 9 y 14 días, fueron marcadamente gliosadas comparadas con las medianamente marcadas en los grupos control e irradiados-dexametasona. Hubo distorsión de la monocapa de Purkinje, con algunas células encontradas en la capa molecular o en la capa de Purkinje, en el grupo irradiado de 5, 9, 14 y 25 días. De los resultados de este estudio, se puede afirmar que la administración de 0,005 ml de dexametasona intraperitonealmente, una hora antes de una exposición a una irradiación, parece proteger el desarrollo del cerebelo de la rata, de lesiones producidas por irradiación.

Gross morphological studies on the effect of cyanide on the developing cerebellum of wistar rat (rattus novegicus).

The teratogenic effect of maternal cyanide consumption on the gross morphology of the post-natal phase of the developing rat cerebellum was studied. Twenty pregnant female rats weighing between 170 g and 190 g were separated into control and experimental groups. The control animals were fed a standard diet of mice cubes, while the experimental animals were fed 500 ppm potassium cyanide, mixed with the standard diet. The diets were fed to the animals and their litters in separate cages and water provided ad libitum during gestation and to the offspring after birth. After birth, the offspring (five per group) of days 1, 9, 14, 21, 28 and 50 were weighed, killed by cervical dislocation and the gross parameters studied. In the experimental animals, no significant differences were observed in the studied parameters between the control and experimental animals on day 1. A significant reduction in body weight was observed on day 14 (P < 0.05). The brain weight was significantly reduced on day 9 (P < 0.05). Similarly, the cerebellar weight was significantly reduced on days 14,21 and 28 (P < 0.05). The maximum vermal length was significantly reduced on day 50 (P < 0.05), and the maximum side-to-side dimension of the cerebellum was also reduced on day 28 (P < 0.05). There was no reduction in the thickness (anteroposterior dimension) of the cerebellum in the experimental group (P > 0.05). From the result, it is inferred that maternal consumption of 500 ppm cyanide causes reduction in the cerebellar weight, vermal length and side-to-side dimension of the developing cerebellum in postnatal life in rats.

Wound healing activities of methanolic extracts Ocimum gratissimum leaf in Wistar rats - a preliminary study.

The wound healing effect of leaf extracts of Ocimum gratissimum was investigated in adult male Wistar rats. Two groups of adult male Wistar rats, average body weight 170g, had a 2cm by 2cm square wound inflicted on the dorsolateral aspect of their trunk with Paniculus Carnosus removed. Experimental group had their wound dressed with methanolic leaf extracts of Ocimum gratissimum while control group had their wounds dressed with normal saline dressing. All animals had wound dressing done every five days; wound dimension measured and, wound morphometry assessed. Wound biopsy was done by random selection in each group on day 10 and the day of complete re-epithelisation. Routine paraffin wax processing was done, slides stained with haematoxylene and eosin for histological assessment of fibroblast count, neovascularisation and granulation tissue profile. The result revealed significant wound contraction (P<0.05) on day 10 in the experimental group (mean 73.40 +/- 3.30)cm2 compared with the control group (mean 53.50 +/- 4.32)cm2. Histology of the healed scar showed non-significant (P>0.05) decrease in the mean fibroblast count forthe experimental group (83.80 +/- 5.70) relative to fibroblast count of 90.20 +/- 17.90 in the control group. The mean blood vessel count was also non-significantly lowered (P>0.05) in the experimental group (9.20 +/- 1.20) relative to the control group (13.40 +/- 2.40). Granulation tissue histology on day 10 showed denser inflammatory infiltrate as reflected by increased cellularity in the control group relative to that of the experimental group which though appeared adequate was not as dense as the control group. Thus we suggest that the methanolic extracts of O. gratissimum could be a potential wound healing agent due to its ability to enhance wound contraction.

A comparative study of the wound healing properties of honey and Ageratum conyzoides.

The present study investigates the wound healing properties of methanolic extracts of Ageratum conyzoides leaves compared with those of honey. Thirty Wistar rats were randomized into 3 groups of 10 animals each. They were fed with standard rat cubes and Tap water weighed and acclimatized to laboratory conditions for one week. Under anesthesia, each animal had the skin of its dorsolateral flank shaved after which an area of the skin was excised. On achieving haemostasis, the wounds were packed with gauze soaked in the appropriate dressing for each group. Measurement of wound size, and wound biopsies were taken on the 10th day post-wound creation. Together with healed wound samples, these were processed for histology. Fibroblast and blood vessel densities per unit area of wound were determined for the healed wound samples. Histologically, the day 10 Ageratum sections showed fewer inflammatory cells compared with similar honey and Control sections. Also, healed scar sections of wounds dressed with the herb extract showed more fibrosis. Honey and Ageratum caused significant greater wound contraction than controls (p = 0.001 and 0.005 respectively). Healed wounds from the Ageratum group had significantly fewer fibroblasts than honey and controls (p = 0.012 and 0.036 respectively).