Use of a caspase multiplexing assay to determine apoptosis in a hypothalamic cell model.

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and decreases waste in costs and reagents. Here we describe optimization of a multiplex assay to assess apoptosis following a palmitic acid (PA) challenge in an in vitro hypothalamic model, using both fluorescent and luminescent based assays to measure viable cell counts and caspase-3/7 activity in a 96-well microtiter plate format. Following PA challenge, viable cells were determined by a resazurin-based fluorescent assay. Caspase-3/7 activity was then determined using a luminogenic substrate, DEVD, and normalized to cell number. This multiplexing assay is a useful technique for determining change in caspase activity following an apoptotic stimulus, such as saturated fatty acid challenge. The saturated fatty acid PA can increase hypothalamic oxidative stress and apoptosis, indicating the potential importance of assays such as that described here in studying the relationship between saturated fatty acids and neuronal function.

Combination of ionising irradiation and hyperthermia activates programmed apoptotic and necrotic cell death pathways in human colorectal carcinoma cells.

PURPOSE: The malignancy of tumor cells can be attenuated by interfering with cell death pathways. Since hyperthermia (HT) is a very potent radiosensitizer, the influence of HT (41.5 °C for 1 hour) alone and in combination with ionising irradiation (X-ray; 5 Gy or 10 Gy) on the form of cell death as well as on the expression of proteins known to be major components in tumor cells' apoptotic and necrotic pathways were examined in colorectal tumor cells. MATERIAL AND METHODS: The expression of proteins was analysed by western blot and the relative activity of caspases-3/7 by fluorescence- based assay. Colony formation was analysed using the clonogenic assay and cell death was determined with annexin V-FITC/propidium iodide staining. RESULTS: Combining X-ray with HT led to similar activation of caspase-3/7 and p53 expression in comparison to irradiation only while the amount of the pro-apoptotic proteins PUMA and Bax was increased in HCT15 and SW480 cells. HT alone or combinations with X-ray further resulted in a temporarily increased level of the anti-apoptotic protein Bcl-2. Irradiation plus HT further led to an up-regulation of IRF-5. The levels of RIP-1, a marker for programmed necrosis, increased in tumor cells which were treated with HT and/or X-ray. Combining 5 Gy irradiation with HT compared to irradiation resulted in a significantly increased number of necrotic tumor cells and in decreased colony formation. CONCLUSION: The combined treatment of colorectal tumor cells with X-ray and HT activates distinct tumor cell pathways and fosters the early appearance of a necrotic tumor cell phenotype.

Induction of apoptosis by immature fruits of Prunus salicina Lindl. cv. Soldam in MDA-MB-231 human breast cancer cells.

The goal of this study was to evaluate the anticancer effect of Prunus salicina Lindl. cv. Soldam at three maturity stages (immature, midmature and mature stages). Previous studies have shown that this fruit (plums) possesses hematopoiesis effects, prevents osteoporosis and has anti-mutagenic effects. An acetone extract of immature P. salicina Lindl. cv. Soldam fruit contained higher levels of total phenolics and condensed tannins than midmature and mature plums. The results showed that an acetone extract of immature plums possesses cytotoxic effects, which are related to the activity of the total polyphenols in the fruits. Apoptosis in MDA-MB-231 cells mediated by the immature plums was associated with an increase in Bax levels and a reduction in Bcl-2 levels and the cleavage of caspase 3, caspase 7, caspase 9 and poly-(ADP-ribose) polymerase. These results indicate that immature fruit of P . salicina Lindl. cv. Soldam can be regarded as a safe and promising new dietary source for decreasing the risk of developing breast cancer.