Blockade of irradiation-induced autophagosome formation impairs proliferation but does not enhance cell death in HCT-116 human colorectal carcinoma cells.

This work was undertaken to gain further information on the molecular mechanisms underlying autophagosome formation and its relation with tumor cell survival in response to radiation in colon cancer. A human colon cancer cell line, HCT-116, was examined with respect to cell survival after blockade of irradiation-induced autophagosome formation by pharmacological interference. Autophagosome formation was confirmed using a kinetic study with incorporated bovine serum albumin gold-conjugate (BSA-Au) analyzed by electron microscopy and an autophagosome-associated LC3B antibody measured by immunofluorescence and Western blotting. Annexin V/PI double staining was used to monitor cell death by apoptosis, and cell cycle profiles by flow cytometry. Ionizing radiation (IR) promoted autophagosome formation in the HCT-116 IR-surviving cells. Pharmacological interference showed that PI3K/Akt and Src were involved in early stages of autophagosome formation. IR alone decreased cell proliferation by arresting cells in the G2/M phase, and pharmacological interference of autophagosome formation decreased proliferation, but did not affect cell survival. Also, our data suggest that decreased proliferation caused by PI3K and Src inhibitors could be through S phase cell cycle delay. Our results clearly indicate that blockade of IR-induced autophagosome formation impairs proliferation but does not enhance cell death in colon cancer cells.

Lysophosphatidic acid induces a migratory phenotype through a crosstalk between RhoA-Rock and Src-FAK signalling in colon cancer cells.

Lysophosphatidic acid (LPA) acts as a potent stimulator of tumorigenesis. Cell-cell adhesion disassembly, actin cytoskeletal alterations, and increased migratory potential are initial steps of colorectal cancer progression. However, the role that LPA plays in these events in this cancer type is still unknown. We explored this question by using Caco-2 cells, as colon cancer model, and treatment with LPA or pretreatment with different cell signalling inhibitors. Changes in the location of adherent junction proteins were examined by immunofluorescence and immunoblotting. The actin cytoskeleton organisation and focal adhesion were analysed by confocal microscopy. Rho-GTPase activation was analysed by the pull-down assay, FAK and Src activation by immunoblotting, and cell migration by the wound healing technique. We show that LPA induced adherent junction disassembly, perijunctional actin cytoskeletal reorganisation, and increased cell migration. These events were dependent on Src, Rho and Rock because their chemical inhibitors PP2, toxin A and Y27632, respectively, abrogated the effects of LPA. Moreover, we showed that Src acts upstream of RhoA in this signalling cascade and that LPA induces focal adhesion formation and FAK redistribution and activation in confluent monolayers. Focal adhesion formation was also observed in the front of migrating cells in response to LPA, and Rock inhibitor abolished this effect. In conclusion, our findings show that LPA modulates adherent junction disassembly, actin cytoskeletal disorganisation, and focal adhesion formation, conferring a migratory phenotype in colon tumour cells. We suggest a functional regulatory cascade that integrates RhoA-Rock and Src-FAK signalling to control these events during colorectal cancer progression.