Intracellular Ca2+ Research for Chemoprevention and Neuroprotection

 

Intracellular Ca2+ is involved in a larger series of cell and physiological processes in health and disease including cell proliferation, cell migration and cell death. Intracellular calcium has been selected by nature as the most pleiotropic and extended second messenger. Calcium ions are not synthesized or destroyed like other messengers. Instead, they are transported up and down electrochemical gradients through a series of transport systems (calcium channels, pumps and co-transporters) located in plasma membrane and endomebranes of the endoplasmic reticulum, mitochondria, Golgi and nuclear envelop. The use of sophisticated approaches of Cell Physiology as Calcium Imaging and Patch Clamp electrophysiology allows to monitor, in real time, changes in the levels of cytosolic and subcellular calcium together with the activity of ion channels. The combination with Molecular Biology approaches including qRT-PCR, western blotting, immunofluorescence, sirRNA and next generation sequenting is enabling the discovery of many important roles of intracellular calcium and calcium transporters in health and disease. We use these approaches to investigate the role of calcium in cancer, aging and Alzheimer┬┤s disease.

Our first goal is to determine the remodelling of subcellular calcium in colorectal cancer and other forms of cancer, its molecular basis, contribution to cancer hallmarks and whether it may provide new opportunities for cancer prevention, diagnostics, prognostics and therapy. For this end we plan to use different cell models of normal cells and tumor cells from colorectal cancer, breast cancer and pancreatic cancer to investigate differences in store-dependent and store-independent Ca2+ entry, store-operated currents, Ca2+ release, Ca2+ store content and transfer of Ca2+ from ER to mitochondria using Ca2+ imaging and patch-clamp electrophysiology. In addition, we plan to investigate the molecular basis of Ca2+ remodelling in cancer by carrying out gene and protein expression analysis of Ca2+ related genes, oncogenes and tumor suppressors using transcriptomics, qRT-PCR, and western blotting in the cell lines mentioned above and in well annotated series of tumor samples from patients obtained from a tumor bank. The actual role of selected genes on tumorogenesis will be tested using knockdown of candidate genes and functional measurements of cancer hallmarks including cell proliferation, migration, invasion and survival in the silenced cells. Finally, based on results, the effects of candidate compounds on Ca2+ remodeling and cancer hallmarks will be tested in cell lines and animal models of cancer. 

Our second goal is to determine the remodelling of subcellular Ca2+ in aging neurons, its molecular basis and whether it may provide novel opportunities for neuroprotection against excitotoxicity and neurodegeneration. For this end, we plan to investigate in detail the remodelling of subcellular Ca2+ in primary cultures of rat hippocampal neurons aged in vitro. Specifically, effects of long-term culture in vitro on Ca2+ entry, Ca2+ release, Ca2+ store content and Ca2+ transfer from ER to mitochondria will be tested using Ca2+ imaging. In addition, we will study the molecular basis of the remodelling by testing the expression of selected molecular players involved in Ca2+ transport, particularly in the transfer from ER to mitochondria (ER-mit coupling) using qRT-PCR, western blotting and quantitative immunofluorescence. Finally, we will extend and validate the results of subcellular Ca2+ remodelling in aging and its molecular players in more physiological models of aging.