The up-regulation of KCC1 gene expression in cervical cancer cells by IGF-II through the ERK1/2MAPK and PI3K/AKT pathways and its significance

Objective: To research the alternation effect of insulin-like growth factors-2 (IGF-II) oil the expression of KCl co-transport-1 (KCC1) in the SiHa cells of cervical cancer, and to explore the activation of ERK1/2MAPK and PI3K/AKT signal transduction pathways during the expression process. Method: To apply semi-quantitative RT-PCR and Western blot analysis to measure changes in mRNA and protein expression of KCC1 after exposure to different concentrations of IGF-II for different time durations in the SiHa cells of cervical cancer. The change in protein expression of the ERK1/2 and AKT pathways is also measured. Furthermore, the protein expression variation in ERK1/2, AKT, and KCC1 is observed after the addition of a specific pathway blocker for the ERK1/2MAPK and PI3K/AKT pathways. Results: The mRNA and protein expression of KCC1 increases dramatically after the application of IGF-II on the SiHa cells, and shows a definite dosage-time dependence relationship. The protein phosphorylation is enhanced in the ERK1/2 and AKT pathways, where the protein activity increases. By adding a specific pathway blocker, the protein activity and phosphorylation of the two pathways are no longer promoted even under the effect of IGF-II. Conclusion: IGF-II can enhance KCC1 gene expression in cervical cancer cells through the ERK1/2MAPK and PI3K/AKT signal transduction pathways.

Insulin-like growth factor II; KCI Co-transport-1; Cervical cancer; RT-PCR, Western blot.

[1] Frield F., Kimura I., Osato T., Ito Y.: "Studies on a new human cell line (SiHa) derived from carcinoma of uterus. I. Its establishment and morphology". Proc. Soc. Exp. Biol. Med., 1970, 135, 543.

[2] Schoell W.M., Janicek M.F., Mirhashemi R.: "Epidemiology and biology of cervical cancer". Semin. Surg. Oncol. , 1999, 16, 203.

[3] Rosenfeld R.G., Roberst C.T.: "The IGF system". Totowa, N.J., Humana Press Inc., 1999.

[4] Mathur S.P., Mathur R.S., Underwood P.B., Kohler M.F., Creasman W.T.: "Circulating levels of insulin-like growth factor-II and IGF-binding protein 3 in cervical cancer". J. Gynecol. Oncol. ,2003, 91, 486.

[5] Serrano M.L., Romero A., Cendales R., Sánchez-Gómez M.,Bravo M.M.: "Serum levels of insulin-like growth factor-I and -II and insulin-like growth factor binding protein 3 in women with squamous intraepithelial lesions and cervical cancer". Biomedica,2006, 26, 258.

[6] Clemmons D.R., Maile L.A.: "Mini review: integral membrane proteins that function coordenately with the insulin-like growth factor I receptor to regulate intracellular signaling. Endocrinology,2003, 144, 1664.

[7] Tanaka Y., Kobayashi H., Suzuki M., Hirashima Y., Kanayama N., Terao T.: "Genetic downregulation of Pregnancy associated plasma protein-A (PAPP-A) by bikunin reduces IGF-I-dependent Akt and ERD1/2 activation and subsequently reduces ovarian cancer cell growth,invasion and metastasis". Int. J. Cancer, 2004, 109, 336.

[8] Schliess F., Schreiber R., Häussinger D.: "Activation of extracellular signal-regulated kinases Erk-1 and Erk-2 by cell swelling in H4ITE hepatoma cells". Biochem. J., 1995, 309, 13.

[9] Shen M.R., Chou C.Y., Browning J.A., Wilkins R.J., Ellory J.C.: "Human cervical cancer cells use Ca2+signaling, protein tyrosine phosphorylation and MAPkinase in regulatory volume decrease".J. Physiol. , 2001, 537, 347.

[10] Rivera C., Voipio J., Thomas-Crusells J., Li H., Emri Z., Sipilä S. et al.: "Mechanism of activity-dependent down regulation of the neuron-specific K-Cl cotransporter KCC2". J. Neurosci, 2004, 24,4683.

[11] Lauf P.K., Adragna N.C.: "KCI cotransport: properies and molecular mechanism". Cell. Physiol. Biochem., 2000, 10, 341.

[12] Bergeron M.J., Gagnon E., Caron L., Isenring P.: "Identification of key functional domains in the C-terminus of the K-CI contransporters". J. Biol. Chem., 2006, 281, 15959.

[13] Shen M.R., Chou C.Y., Hsu K.F., Hsu Y.M., Chiu W.T., Tang M.J., et al.: "KCl cotransport is an important modulator of human cervical cancer growth and invasion". J. Biol. Chem., 2003, 278, 39941.

[14] Lang F, Föller M., Lang K.S., Lang P.A., Ritter M., Gulbins E. et al.: "Ion channels in cell proliferation and apoptotic cell death".J. Membrane Biol., 2005, 205, 147.

[15] Shen M.R., Wilkins R.J., Chou C.Y., Ellory J.C.: "Anion exchanger isoform 2 operates in parallel with Nat/Ht exchanger isoform 1 during regulatory volume decrease of human cervical cancer cells". FEBS Lett. , 2002, 512, 52.

[16] Shen M.R., Chou C.Y., Ellory J.C.: "Swelling activated taurine and K+transport in human cervical cancer cells: association with cycle progression". Pflugers Arch. , 2001, 441, 787.

[17] Shen M.R., Chou C.Y., Ellory J.C.: "Volume-sensitive KCI cotransport associated with human cervical carcinogenesis". Pflugers Arch. , 2000, 440, 751.

[18] Shen M.R., Lin A.C., Hsu Y.M., Chang T.J., Tang M.J., Alper S.L. et al.: "Insulin-like growth factor 1 stimulates KCl co-transport, which is necessary for invasion and proliferation of cervical cancer and ovarian cancer cells". J. Biol. Chem. , 2004, 279, 40017.

[19] Shen M.R., Chou C.Y., Hsu K.F., Hsu Y.M., Chiu W.T., Tang M.J., et al.: "KCl cotransport is an important modulator of human cervical cancer growth and invasion". J. Biol. Chem. , 2003, 278, 39941.