Antiproliferative effect of growth hormone-releasing hormone (GHRH) antagonist on ovarian cancer cells through the EGFR-Akt pathway

Abstract

Background: Antagonists of growth hormone-releasing hormone (GHRH) are being developed for the treatment of various human cancers.

Methods: MTT assay was used to test the proliferation of SKOV3 and CaOV3. The splice variant expression of GHRH receptors was examined by RT-PCR. The expression of protein in signal pathway was examined by Western blotting. siRNA was used to block the effect of EGFR.

Results: In this study, we investigated the effects of a new GHRH antagonist JMR-132, in ovarian cancer cell lines SKOV3 and CaOV3 expressing splice variant (SV)1 of GHRH receptors. MTT assay showed that JMR-132 had strong antiproliferative effects on SKOV3 and CaOV3 cells in both a time-dependent and dose-dependent fashion. JMR-132 also induced the activation and increased cleaved caspase3 in a time- and dose-dependent manner in both cell lines. In addition, JMR-132 treatments decreased significantly the epidermal growth factor receptor (EGFR) level and the phosphorylation of Akt (p-Akt), suggesting that JMR-132 inhibits the EGFR-Akt pathway in ovarian cancer cells. More importantly, treatment of SKOV3 and CaOV3 cells with 100 nM JMR-132 attenuated proliferation and the antiapoptotic effect induced by EGF in both cell lines. After the knockdown of the expression of EGFR by siRNA, the antiproliferative effect of JMR-132 was abolished in SKOV3 and CaOV3 cells.

Conclusions: The present study demonstrates that the inhibitory effect of the GHRH antagonist JMR-132 on proliferation is due, in part, to an interference with the EGFR-Akt pathway in ovarian cancer cells.

Figure 1

The expression of mRNA for GHRHR SV1 in SKOV3 and CaOV3 human ovarian cancer cells. The prostate cancer cell line LNCaP was used as a positive control. Human GAPDH was used as an internal control.

Figure 2

Antiproliferative effect of JMR-132 on SKOV3 and CAOV3 cells. SKOV3 (2000/well) and CaOV3 (3000/well) were treated with JMR-132 at a different dose (25, 50, 100 and 200 nM) per day for 2 days (A), (C) and with 100 nM JMR-132 per day for different periods of time (24, 48, 72 and 96 h) (B) (D). MTT assay showed that proliferation was inhibited in a dose (A) and time (B) dependent manner after JMR-132 treatment. * P < 0.05 compared to the no treatment group. The expression of cleaved caspase3 increased in a dose (C) and time (D) dependent manner. The results indicated that JMR-132 treatment induced apoptosis in these cells.

Figure 3

The expression of EGFR in SKOV3 and CaOV3 cells decreased after JMR-132 treatment. The protein level of EGFR decreased after treatment with 100 nM JMR-132 per day for 2 and 4 days. β-actin was used as an internal control. The data are from one experiment and are representative of the three separate experiments.

Figure 4

JMR-132 attenuated the effect of EGF-induced p-Akt activation. A. p-Akt was activated after EGF (10 ng/ul) treatment without changes in Akt. β-actin was used as an internal control. B. p-Akt expression decreased after treatment with 100 nM JMR-132 per day for 2 and 4 days without changes in Akt level. C. JMR-132 attenuated the effect of EGF-induced p-Akt expression. JMR-132 (100 nM) and PI3K specific inhibitor LY294002 (10 nM) were used separately to continuously treat SKOV3 and CaOV3 cells for 2 days. PI3K specific inhibitor LY294002 was used as a positive control. β-actin was used as an internal control. EGF was added 15 min prior to harvesting.

Figure 5

JMR-132 abolished the pro-proliferative and anti-apoptotic effect of EGF. A. Using the MTT assay, it was determined that the growth of SKOV3 and CaOV3 cells was significantly inhibited after continuous treatment with 100 nM JMR-132 for 2 days in combination with EGF (10 μg/ml). Letters (a, b, c) between pairs indicate significant differences (P < 0.05). B. The expression of cleaved caspase3 increased during treatment with 100 nM JMR-132 per day for 2 days in combination with EGF (10 μg/ml). β-actin was used as an internal control. EGF was added 15 min prior to harvesting in (A) and (B).

Figure 6

No changes were detected after transfection with EGFR siRNA, either with or without JMR-132 treatment. A. EGFR was knocked down after transfection with EGFR siRNA for various periods of time. After transfection with 100 nM EGFR siRNA for 48 hours, we reseeded the cells for the MTT assay. Pretreatment: after cell reseeding; 48 and 96 hours: treatment with 100 nM JMR-132 per day for 2 and 4 days after cell reseeding. The EGFR expression was still lower when compared to the cells transfected with 100 nM control siRNA. B. No changes were found by the MTT assay after transfection with EGFR siRNA, with or without JMR-132 treatment. The cells (SKOV3 and CaOV3: 4000/well) were reseeded in a 96-well plate after transfection with control siRNA or EGFR siRNA for 2 days. After 2 and 4 days of treatment with 100 nM JMR-132, the MTT assay was performed. Letters (a, b, c) indicate significant differences (P < 0.05) between pairs. C. No changes were detected in cleaved caspase3 expression after transfection with EGFR siRNA, either with or without JMR-132 treatment in SKOV3 cells. After transfection with 100 nM EGFR siRNA for 48 hours, the SKOV3 cells were treated with 100 nM JMR-132 per day for 2 days. β-actin was used as an internal control.