Selenite-induced Survival of HuH7 Hepatoma Cells Involves Activation of Focal Adhesion Kinase-Phosphatidylinositol 3-Kinase-Akt Pathway and Rac1

Abstract

Selenium has been shown to sustain the growth of selected human hepatocellular carcinoma cell lines under serum-free conditions, but the detailed mechanism remained undetermined. In the present study, the molecular mechanism(s) involving sodium selenite (Na 2 SO 3 , Se) as a survival agent were determined. Selenite not only protects HuH7 cells from serum deprivation-induced apoptosis, it also supports its long-term growth in sodium selenite (10 ؊7 M) supplemented serumfree medium. The anti-apoptotic effect correlates with activation of focal adhesion kinase and the phosphatidylinositol 3-kinase (PI3K)-Akt kinase pathway. Using HuH7 cells stably transfected with a constitutively active Akt kinase and PI3K inhibitor LY294002, seleniteinduced cell survival was shown to be PI3K-Akt-dependent. Parallel changes included a significant reduction in the intracellular reactive oxygen species content, the reversal of DNA fragmentation, and the suppression of caspase and apoptosis signal-regulating kinase 1 activities. HuH7 cells stably expressing a Rac1 mutant N17 (Rac1 N17 -HuH7) are refractory to selenite treatment. In these cells selenite supplement neither triggers Akt activation nor supports cell proliferation. Participation of Rac1 activation in this event is supported by the fact that selenite treatment drastically enhanced activation of Rac1. The exact link between selenite treatment, Rac1 activation, and activation of the focal adhesion kinase-PI 3-kinase, however, remains to be characterized. The mitogenic signaling mediated by selenite may involve unconventional growth stimuli including higher glutathione peroxidase 1 activity and higher transcription levels of selenoprotein P. The selenium-HuH7 system we have established thus provides a unique tool that will allow the biological role of selenite in growth regulation of hepatocytes to be studied in detail.The essential biological trace element, selenium, plays a vital part in many metabolic functions (1, 2). Selenium in selenoproteins is in the form of selenocysteine, which is synthesized cotranslationally from serine and selenide as selenocysteine by a series of enzymatic reactions dictated by the UGA codon (3) and SECIS sequences (4, 5). A wide spectrum of biological functions is performed by the selenoprotein family, ranging from antioxidant protection and thyroid hormone metabolism to proper reproductive performance. Selenoproteins are crucial to many cellular functions as illustrated by the fact that overexpression of glutathione peroxidase 1 (GPX1) 1 (6 -8) and thioredoxin reductase (9, 10) have been reported to inhibit apoptosis and selenoprotein P seems to be a survival factor for neurons (11,12).Extensive effort has been devoted to the study of the chemopreventive action of selenium in the recent years because cumulative evidence from both experimental and epidemiological approaches has linked low plasma levels of selenium with a high incidence of cancer in humans (13-15). Encouraging results from several large clinical trials demonstrated that selenium...

Identification of SOX4 target genes using phylogenetic footprinting-based prediction from expression microarrays suggests that overexpression of SOX4 potentiates metastasis in hepatocellular carcinoma

“…Downregulation of SOX4 did not significantly affect cell growth (Figure 2c) but rather induced distinct morphological changes in the HCC cells (Figure 2d). Five to seven days after shSOX4 lentiviral infection, HuH7 cells exhibited a less-transformed pavement-like cell arrangement (Lee et al, 2003) whereas giant fused cells were noticed in the SK-Hep1 and Mahlavu cultures. Upon close examination with phallodine staining, F-actin positive membrane protrusions were significantly reduced in addition to a loss of F-actin organization in the shSOX4-treated cells (Figure 2e).…”

“…Antibodies, immunoblotting, immunostaining and staining for F-actin Immunoblotting was performed as described previously (Lee et al, 2003). Nuclear and cytosolic lysates were prepared using a CelLytic Nuclear Extraction System Kit (Sigma).…”

Type 3 repeat/C-terminal domain of thrombospondin-1 triggers caspase-independent cell death through CD47/αvβ3 in promyelocytic leukemia NB4 cells

“…The lower level of ROS observed at day 3 was very likely resulting from ROS leakage due to membrane alterations. Pretreatment of the cells with selenium, known to reduce oxidative stress, or ebselen, a selenoorganic compound that converts H 2 O 2 to H 2 O, 43 showed no inhibitory effect, indicating that mitochondria-derived ROS were likely not involved in the cell death process induced by T3C1 ( Figure 5C). …”

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“…Selenium, an essential biological trace element, has been shown to modulate functions of many regulatory proteins involved in signal transduction, and to affect a variety of cellular activities including cell growth, survival and death [1,2] . The relationship between selenium and signal molecules has not been well elucidated.…”