Expression and significance of HERG (KCNH2) potassium channels in the regulation of MDA-MB-435S melanoma cell proliferation and migration.

The human ether-a-go-go related gene (HERG) potassium channel has elicited intense scientific interest due to its role in cardiac repolarization and its association with arrhythmia and sudden cardiac death. Increasing evidence indicates the involvement of HERG channels in the pathophysiology of cancer. In the present study we investigated the expression of HERG protein in MDA-MB-435S melanoma cells, and its importance in regulating cell proliferation and migration. Our results showed that HERG was expressed on protein and mRNA levels in MDA-MB-435S melanoma cells. In these cells blockade of HERG channels with the HERG blockers E 4301 or cisapride attenuated both proliferation and migration of the cells. Activation of HERG with PD118057 stimulated cell migration. Furthermore, HERG small interfering (si) RNA attenuated the proliferation and migration of the cells. Incubation of MDA-MB-435S cells with E 4301 decreased the phosphorylation of mitogen-activated protein (MAP) kinase and the expression of the c-fos transcription factor. In control experiments, overexpression of HERG channels in HEK-293 cells dramatically increased the proliferation and migration of the cells and blocking HERG in these cells attenuated both proliferation and migration. Our results indicate that MDA-MB-435S cells express HERG channels and blockade of HERG results in the attenuation of both proliferation and migration by a mechanism dependent, at least in part, on an inhibition of the MAP kinase/c-fos pathway.

Regulation of HERG (KCNH2) potassium channel surface expression by diacylglycerol.

The HERG (KCNH2) channel is a voltage-sensitive potassium channel mainly expressed in cardiac tissue, but has also been identified in other tissues like neuronal and smooth muscle tissue, and in various tumours and tumour cell lines. The function of HERG has been extensively studied, but it is still not clear what mechanisms regulate the surface expression of the channel. In the present report, using human embryonic kidney cells stably expressing HERG, we show that diacylglycerol potently inhibits the HERG current. This is mediated by a protein kinase C-evoked endocytosis of the channel protein, and is dependent on the dynein-dynamin complex. The HERG protein was found to be located only in early endosomes and not lysosomes. Thus, diacylglycerol is an important lipid participating in the regulation of HERG surface expression and function.

Phorbol 12-myristate 13-acetate inhibits FRO anaplastic human thyroid cancer cell proliferation by inducing cell cycle arrest in G1/S phase: evidence for an effect mediated by PKCdelta.

Phorbol 12-myristate 13-acetate (PMA) is known to affect a variety of cellular processes, including cell proliferation, differentiation, and migration. PMA has been shown to promote antiproliferative and antimigratory effects in many types of cancer cells. Our findings show that PMA induced a strong antiproliferative effect in two anaplastic (FRO and ARO) and one follicular (ML-1) thyroid cancer cell lines, and increased the fraction of FRO cells in G1 phase of the cell cycle. The fractions in the S and G2 phases were decreased. Moreover, PMA evoked a significant increase in the levels of the cell cycle regulators p21Waf1/Cip1 and p27Kip1. The levels of cyclin D3 and the cyclin-dependent kinases cdk4 and cdk6 decreased, as did the phosphorylation of the Rb-protein. PMA did not induce apoptosis. PMA stimulated the translocation of protein kinase C (PKC) alpha, betaI and delta isoforms to the cell membrane. PKCdelta small interfering RNA attenuated the PMA-induced antiproliferative effect and prevented the upregulation of p21Waf1/Cip1 and p27Kip1. Prolonged stimulation with PMA decreased the phosphorylation of mitogen-activated protein (MAP) kinase. PMA also decreased the phosphorylation of Akt and evoked a biphasic change in the phosphorylation of the forkhead box class-O protein (FOXO): an increase in phosphorylation, followed by a dephosphorylation. In addition, PMA inhibited FRO, ARO and ML-1 cell migration toward serum. The inactive phorbol ester analog 4alpha-phorbol and the diacylglycerol analog 1,2-dioctanoyl-sn-glycerol were without an effect on proliferation and migration. The results indicate that PMA is an effective inhibitor of thyroid cancer cell proliferation and migration by a mechanism involving PKC-MAP kinase/Akt and FOXO signaling.

Antiproliferative effect of sphingosylphosphorylcholine in thyroid FRO cancer cells mediated by cell cycle arrest in the G2/M phase.

Among the group of bioactive sphingolipids, sphingosylphosphorylcholine (SPC) has been known to induce both antiproliferative and proliferative effects depending on cell type. In the present investigation we show that SPC (1-10 microM) reduced the proliferation of FRO cells (an anaplastic thyroid carcinoma cell line) in a concentration dependent manner. The effect was pertussis toxin insensitive, and independent of phospholipase C, protein kinase C, p38 kinase, or jun kinase. In addition to inhibiting the migration of FRO cells, application of SPC induced a rapid (<10 min) rounding of the cells, which was dependent on extracellular sodium. However, DAPI staining and caspase-3 analysis could not reveal any apoptotic effects of SPC. Furthermore, when cells treated with SPC for 24h were washed and replated, they continued to grow, albeit somewhat slower than control cells. Flow cytometry analysis revealed a significant increase in the population of cells in the G2-M phase, and a reduction in S phase. SPC reduced the phosphorylation of Akt with about 50% and evoked a substantial decrease in the amount of phosphorylated mitogen-activated protein (MAP) kinase. In cells treated with the PI3 kinase inhibitor wortmannin, both migration and proliferation were inhibited, as well as the amount of phosphorylated MAP kinase. Treatment of the cells with either SPC or wortmannin increased the levels of p21, but decreased that of cyclin B1 and Cdc2. Taken together, SPC is an effective suppressor of thyroid cancer cell proliferation and migration, and this effect is, in part, mediated by inhibition of both the PI3K-Akt and the MAP kinase signalling pathways.

Sphingosylphosphorylcholine enhances calcium entry in thyroid FRO cells by a mechanism dependent on protein kinase C.

Several sphingolipid derivatives, including sphingosylphosphorylcholine (SPC), regulate a multitude of biological processes. In the present study we show that both human thyroid cancer cells (FRO cells) and normal human thyroid cells express G protein-coupled receptor 4 (GPR4) and ovarian cancer G protein-coupled receptor 1 (OGR1), putative SPC-specific receptors. In FRO cells SPC evoked a concentration-dependent increase in intracellular free calcium concentration ([Ca2+]i) in a calcium containing, but not in a calcium-free buffer. Sphingosine 1-phosphate (S1P) evoked an increase in [Ca2+]i in both a calcium containing and a calcium-free buffer. The phospholipase C (PLC) inhibitor U 73122 potently attenuated the effect of SPC, suggesting that effects of SPC were mediated by a G protein coupled receptor. Overnight pretreatment of the cells with pertussis toxin did not affect the SPC-evoked response. Interestingly, SPC did not evoke an increase in inositol phosphates, although S1P did so. Furthermore, in cells pretreated with thapsigargin to deplete intracellular calcium stores, SPC still evoked an increase in [Ca2+]i, suggesting that SPC mainly evoked entry of extracellular calcium. When the cells were pretreated with the protein kinase C (PKC) inhibitor GF 109203X, or when the cells were pretreated with PMA for 24 h, the SPC-evoked calcium entry was attenuated. Thus, the SPC-evoked calcium entry was apparently dependent on PKC. In sharp contrast, the increase in [Ca2+]i evoked by S1P was not sensitive to GF 109203X. Furthermore, the calcium entry evoked by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol was not inhibited by GF 109203X. In addition, SPC decreased the incorporation of 3H-thymidine in a concentration-dependent manner in FRO cells. Taken together, SPC may be an important factor regulating thyroid cancer cell function.