Inhibition of LKB1 abrogates honokiol mediated modulation of AMPK and inhibition of migration and invasion of breast cancer cells The tumor suppressor LKB1 is surely an evolutionarily conserved serine/threonine protein kinase that has a broad array of cellular functions, which include tumor suppression, cell polarity, cell cycle regulation, and promotion of apoptosis. LKB1 has just lately been identified being a crucial upstream kinase for AMPK, regulating its activity. Intriguingly, we observed that hono kiol increases expression of tumor suppressor LKB1 in MCF7 and MDA MB 231 cells, using a major boost at one hour of treatment with maximal expression at 24 hrs in MCF7 cells and at four hrs in MDA MB 231 cells. Variable expression of LKB1 in MDA MB 231 breast cancer cells has become reported.
We a short while ago procured MDA MB 231 cells from different established breast cancer exploration laboratories and analyzed the expression and functional status of LKB1. Our data unequivocally showed selleck chemical the presence of functional LKB1 in MDA MB 231 cells. Human LKB1 is each nuclear and cytoplasmic, but a mutant of LKB1 lacking the nuclear localization signal even now retains the ability to suppress cell growth, suggesting that the cytosolic pool of LKB1 plays a vital part in med iating its tumor suppressor properties. STRAD protein has been shown to kind a complex by which STRAD activates LKB1, resulting in cytoplasmic translocation of LKB1. We investigated the effect of honokiol over the formation of the LKB1 STRAD complex in breast cancer cells. To address this question, breast cancer cells had been taken care of with honokiol followed by immunoprecipitation with LKB1 antibodies.
Immunoprecipitated protein complexes have been analyzed for your presence of STRAD by utilizing Western blot ana lysis. Larger amounts of STRAD immunoprecipitated EX-527 with LKB1 in the presence of honokiol indicated increased formation of your LKB1 STRAD complex. Immunostaining of honokiol taken care of MCF7 and MDA MB 231 cells uncovered that honokiol treatment increases cytoplasmic accumulation of LKB1. LKB1 was localized predominantly during the nucleus in untreated breast cancer cells, whilst cytoplasmic LKB1 expression was also detected. Manage experiments with 2nd ary antibody gave an exceptionally faint background staining that was distributed uniformly throughout the cells, irrespective of the treatment method.
Stu dies on the subcellular localization of LKB1 have indi cated a wide range of localization patterns. Mouse LKB1 was located to be predominantly nuclear, whereas Caenorhabditis elegans PAR 4 and Xenopus XEEK1 had been detected exclusively inside the cytoplasm. Human LKB1 has been detected to become the two nuclear and cytoplasmic in numerous cell styles. Although LKB1 expression is solely cytoplasmic in lung and pan creatic cancer, gastrointestinal hamartomatous polyps from Peutz Jeghers syndrome patients, head and neck squamous cell carcinoma, invasive lobular breast carcinoma, and reliable papillary ductal carcinoma in situ breast cancer display both cytoplasmic and nuclear LKB1 expression.