Cisplatin and PI3kinase inhibition decrease invasion and migration of human ovarian carcinoma cells and regulate matrix-metalloproteinase expression

Abstract

Targeting of the PI3K (phosphoinositide3-kinase)/Akt/mTOR pathway in human ovarian cancer cells is a promising novel therapeutic strategy. We investigated the effects of cisplatin and the PI3K inhibitor LY294002 on invasion, migration and the expression of essential matrix metalloproteinases (MMPs) in ovarian cancer cells. SKOV3, OVCAR5 and IGROV1 human ovarian cancer cell lines were treated with cisplatin, LY294002 and a combination of both drugs. Invasion and migration of treated cells was assessed using Matrigel and uncoated PET membrane assays. Expression levels of pro-MMP2, MMP2, TIMP1, TIMP2 and MT1-MMP were determined using Western Blotting. Gel zymography was used to quantitate the functional levels of active MMP2. All three cell lines showed significantly reduced invasion and migration after treatment with cisplatin, LY294002, and the combination of both drugs compared to untreated controls. In SKOV3 cells, cisplatin alone and in combination with LY294002 resulted in a 6.3 and 7.1-fold reduction in the total amount of activated MMP2. TIMP1 expression decreased by 5.0, 6.6 and 28.4-fold with cisplatin, LY294002 and the combination respectively (P < 0.05). In contrast, only cisplatin and the combination of both drugs resulted in a significant, 3.7 and 5.1-fold reduction in the level of TIMP2. Expression levels of MT1-MMP remained unchanged. These observations were corroborated in IGROV1 cell lines that showed similar changes of activated MMP2 and TIMP2 expression, but no significant decrease in TIMP1 levels. Our data suggests that inhibition of ovarian cancer cell motility is mediated via down-regulation of activated MMP2, TIMP1 and TIMP2 expression under these treatment conditions.

Figure 1

Migration and invasion of cisplatin (Cis), LY294002 (LY) and combination treated SKOV3, IGROV1 and OVCAR5 cells. (a) Photomicrograph of SKOV3 cells invading through Matrigel-coated membranes stained with Crystal violet. (b) Mean number of invaded cells normalized to their respective viability ratio ± SEM under different treatment conditions. (c) Mean number of migrated cells normalized to their respective viability ratio ± SEM under different treatment conditions. * p < 0.05, ** p < 0.01, *** p < 0.001 by 1 way ANOVA. (d) Ratio of viable over total tumor cells as determined using a Vi-CELL Cell Viability Analyzer at 24 and 48 hours of incubation under treatment conditions.

Figure 2

Inhibition of pAkt signaling by LY294002 in SKOV3, IGROV1 and OVCAR5 cells. (a) Western blot analysis of control and LY294002 treated SKOV3, IGROV1 and OVCAR5 cell lysates using antibodies against pAkt, Akt and its downstream effectors pS6, S6 and p4E-BP1. β-actin was used to control for equal protein loading. (b) The relative amounts of activated pAkt and pS6 protein were determined by densitometric analysis of their respective band intensities in each cell line.

Figure 3

MMP2 activity by gelatin zymography of conditioned medium from treated SKOV3 and IGROV1 cells. (a) Gelatin zymography for MMP2. The upper band corresponds to the 72 kDa proform MMP2 gelatinase, the lower band represents the 62 kDa activated form. (b), (c) Densitometric analysis of pro-MMP2 and MMP2 activity. The value of MMP2 and pro-MMP2 activity is shown as percent of untreated control cells. * p < 0.05, ** p < 0.01, *** p < 0.001 by 1 way ANOVA.

Figure 4

Expression of TIMP1 and TIMP2 in SKOV3 and IGROV1 cells treated with cisplatin (Cis), LY294002 (LY) or combination. (a) Western blot analysis of conditioned media from control and treated SKOV3 and IGROV1. (b), (c) Densitometric analysis of TIMP1 and TIMP2 band intensities. The value of TIMP1 and TIMP2 protein levels is shown as percent of control cells. * p < 0.05, ** p < 0.01, *** p < 0.001 by 1 way ANOVA.

Figure 5

MT1-MMP levels in SKOV3 and IGROV1 cells treated with cisplatin (Cis), LY294002 (LY) or the combination express similar levels of MT1-MMP. (a) Western blot of cell lysates from treated SKOV3 and IGROV1 cells using antibodies against MT1-MMP. (b) Densitometric analysis of MT1-MMP band intensities. The value of MT1-MMP protein levels in treated SKOV3 and IGROV1 cells is shown as percent of the control value.