Figure 4.

Bacopa monnieri (BM) a known inducer of CaMK2A phosphorylation generates glioblastoma cytotoxicity. (A) BM extract, an inducer of CaMK2A phosphorylation, was administered to LN229 glioblastoma tumor cell line cultures in various doses and monitored at various time points. Human normal radial glial cells (SVG) were used as normal controls. Top insets in middle and last panels of LN229 shows that BM treatments (100 μg/ml and 150 μg/ml), within 12 h, induced phased lucent enlarged vacuoles in cells, characteristic of macropinosomes, also see Supplementary Figure S5A for confirmation of macropinocytotic vacuoles. Second inset in the middle panel of LN229 shows Dextran TMR (red, which is uptaken by macropinosomes and released into the cytoplasm) filled, rounded and swelled tumor cell with squashed nucleus (white, due to raised hydrostatic pressure of intracellular fluid) in 24 h post treatment, please see Supplementary Figure S5B for more detailed images. Last inset shows loss of cell actin cables (rhodamine phallodin staining shown in cyan) due to cell swelling. The comparative analysis of main panels on the basis of morphology and F-actin staining suggests no cytotoxicity to normal cells, however, LN229 cells showed cell rounding (cyan arrows), cell swelling, nuclear compression (magenta arrows) and cell necrosis (red arrows), post 24 h of treatment. A dose of 150 μg/ml showed SVG differentiated phenotype, hence the therapeutic dose was determined to be 100 μg/ml. (B,C) Calcein-AM (green, live cell marker probe)/EtBr (red, damaged and dead cell marker dye) based cytotoxicity and viability analysis over various time points (12, 24 and 48 h) post BM treatment at 0, 100 and 150 μg/ml showed specific cytotoxic effects on LN229 glioblastoma cell lines vs. the normal cells (SVG and HaCaT). ***p < 0.001. Error bar = SD The analysis is representative of three independent experiments.