Activation of the PKC pathway stimulates ovarian cancer cell proliferation, migration, and expression of MMP7 and MMP10

Abstract

Postmenopausal women are at a higher risk of ovarian cancer due, in part, to increased levels of gonadotropins such as luteinizing hormone (LH). Gonadotropins and other stimuli are capable of activating two pathways, PKA and PKC, that are altered in ovarian cancer. To determine the role of LH on ovarian cancer, we explored the effects of human chorionic gonadotropin (hCG), an LH mimic, and an activator of the PKC pathway, phorbol-12-myristate 13-acetate (PMA), on ovarian cancer cell-cycle kinetics and apoptosis in Ovcar3 cells. PMA treatment increased cells in the S phase of the cell cycle and initially increased apoptosis after 4 h before diminishing apoptosis after 8 h. Treatment of ovarian cancer cells with hCG had no effect on these parameters. The PKC pathway is known to differentially regulate matrix metalloproteinase (MMP) expression. Results showed that ovarian cancer cells treated with PMA increased MMP7 and MMP10 mRNA levels after 8 h of treatment, and expression remained high after 12 h before decreasing at 24 h. The mRNA expression of extracellular matrix metalloproteinase inducer (BSG), an activator of MMPs, was unaffected by PMA. Due to the role that MMPs play in migration, we investigated the effect of PMA activation of MMPs on ovarian cancer cell migration. The use of the MMP inhibitor GM6001 blocked the increased migratory effects of PMA on ovarian cancer cells. Together, these studies show that activating the PKC pathway causes significant changes in cell cycle kinetics and selective expression of MMPs that are involved in enhancing ovarian cancer cell proliferation and migration.

Keywords: MMPs; cancer; gene expression; migration; ovary.

FIG. 1

Ovcar3 cell cycle kinetics after hCG or PMA treatment. Ovcar3 cells were serum starved for 24 h and treated with 1 IU hCG or 20 nM PMA for an additional 24 h. Percentage of the cells in (A) the G₀/G₁stage of the cell cycle, (B) the G₂/M stage of the cell cycle, or (C) the S phase of the cell cycle. Results are the means ± SEM for at least three separate measurements from three individual experiments. Bars that do not share a letter designation are significantly different (P < 0.05).

FIG. 2

Apoptosis of Ovcar3 cells after hCG or PMA treatment. Ovcar3 cells were serum starved for 24 h and treated with vehicle control (DMSO), 20 nM PMA, or 1 IU hCG for (A) 4 h, (B) 8 h, or (C) 12 h. Results are the means ± SEM of at least three separate measurements from three individual experiments. White bars represent viable cells; black bars represent cells undergoing early and late apoptosis as well as those that are dead. Bars that do not share a letter or number designation are significantly different (P < 0.05).

FIG. 3

Treatment with hCG increases cAMP in Ovcar3 cells. Cells were serum starved for 1 h and treated for 20 min with vehicle control, hCG, or FSK. Human CG increased cAMP activity 4-fold, and FSK increased cAMP by 9-fold. Results are the means ± SEM for three to five measurements from separate Ovcar3 cell cultures. A one-way ANOVA was performed; bars that do not share a letter designation are significantly different within a treatment group (P < 0.05).

FIG. 4

MMP7, MMP10, MMP9, and BSG mRNA expression after temporal PMA treatments. Cells were serum starved for 24 h and treated for an additional 4, 8, 12, or 24 h with vehicle control (DMSO) or 20 nM PMA. Messenger RNA expression was analyzed by real-time PCR at the indicated time points for (A) MMP7, (B) MMP10, (C) MMP9, and (D) BSG (i.e., Basigin) expression. Results are the means ± SEM for three individual measurements. A two-way ANOVA was performed; bars that do not share a letter designation are significantly different within a treatment group (P < 0.05).

FIG. 5

Ovcar3 cell migration with PMA is prevented by MMP inhibitors. Cells were treated with vehicle control (DMSO) or 20 nM PMA in combination with or without 25 μM GM6001, 35 μM GM6001, an MMP2/9 inhibitor, or both MMP inhibitors. Migration was assessed after 24 h of incubation. A two-way ANOVA was performed; bars that do not share a letter designation are significantly different within a treatment group (P < 0.05).