Characterisation of the in vitro effects of Asiatic acid in Glioblastoma

Glioma arises from glial cells in the brain and is the most common intracranial neoplasm of the central nervous system. It is known that tumour masses are hypoxic and a drop in the partial pressure of oxygen activates hypoxia inducible factor that plays an important role in tumour growth by controlling the transcription of genes responsible for cell proliferation, angiogenesis and energy metabolism. Asiatic acid, a pentacyclic triterpenoid extracted from Centella asiatica, has shown cytotoxicity against many cancer cell lines. To determine its anti-cancer effects under hypoxia, we investigated the effects of asiatic acid on U87-MG cell line, a grade IV glioblastoma cell line under normoxia and 1% & 5% hypoxia. Asiatic acid efficacy on glioblastoma cells was assessed using fluorescent dyes along with flow cytometric and microscopic analysis. A major problem associated with the administration of asiatic acid is low aqueous solubility thus hindering dissolution and causing decreased bioavailability. Thus we formulated asiatic acid-loaded poly-ε-caprolactone nanoparticles for sustained and localised delivery in glioblastoma cells in vitro. Asiatic acid nanoparticles were prepared using solvent displacement method and showed an average diameter of 150nm, average negative surface charge of -20mV and were stable for a minimum of 60 days at 4°C. Results showed no drug release or nanoparticle degradation in the absence of lipase in aqueous media. Nanoparticles were stable in PBS and presence of lipase enhanced drug release. Asiatic acid reduced cell viability in U87-MG cell line in a time and concentration-dependent manner. Flow cytometric analysis revealed a large number of cells undergoing cell death via apoptosis under normoxia. A significant delay in wound healing was observed with asiatic acid treatment under normoxia and a only partial closure of the wound was observed following 18 hours of incubation. Under hypoxia, a significantly large number of cells underwent apoptosis in comparison to the chemotherapy drug cisplatin. We did not observe any significant effects on cell proliferation or cell cycle

Pharmacological Effects of Asiatic acid in Glioblastoma Cells under Hypoxia

Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumor in adults. Despite current treatment options including surgery followed by radiation and chemotherapy with temozolomide (TMZ) and cisplatin, the median survival rate remains low (<16 months). Combined with increasing drug resistance and the inability of some compounds to cross the blood brain barrier (BBB), novel compounds are being sought for the treatment of this disease. Here, we aimed to examine the pharmacological effect of Asiatic acid (AA) in glioblastoma under hypoxia. To investigate the effects of AA on cell viability, proliferation, apoptosis and wound healing, SVG p12 fetal glia and U87-MG grade IV glioblastoma cells were cultured under normoxic (21% O2) and hypoxic (1% O2) conditions. In normoxia, AA reduced cell viability in U87-MG cells in a time and concentration-dependent manner. A significant decrease in viability, compared to cisplatin, was observed following 2hrs of AA treatment with no significant changes in cell proliferation or cell cycle progression observed. Under hypoxia, a significantly greater number of cells underwent apoptosis in comparison to cisplatin. While cisplatin showed a reduction in wound healing in normoxia, a significantly greater reduction was observed following AA treatment. An overall reduction in wound healing was observed under hypoxia. The results of this study show that AA has cytotoxic effects on glioma cell lines and has the potential to become an alternative treatment for glioblastoma

A high-throughput pipeline for validation of antibodies

Western blotting (WB) is widely used to test antibody specificity, but the assay has low throughput and precision. Here we used preparative gel electrophoresis to develop a capture format for WB. Fractions with soluble, size-separated proteins facilitated parallel readout with antibody arrays, shotgun mass spectrometry (MS) and immunoprecipitation followed by MS (IP-MS). This pipeline provided the means for large-scale implementation of antibody validation concepts proposed by an international working group on antibody validation (IWGAV).Western blotting (WB) is widely used to test antibody specificity, but the assay has low throughput and precision. Here we used preparative gel electrophoresis to develop a capture format for WB. Fractions with soluble, size-separated proteins facilitated parallel readout with antibody arrays, shotgun mass spectrometry (MS) and immunoprecipitation followed by MS (IP-MS). This pipeline provided the means for large-scale implementation of antibody validation concepts proposed by an international working group on antibody validation (IWGAV)