Administration of glucocorticoids to ovarian cancer patients is associated with expression of the anti-apoptotic genes SGK1 and MKP1/DUSP1 in ovarian tissues

Abstract

Purpose: To prevent chemotherapy-related side effects, synthetic glucocorticoids, for example, dexamethasone, are routinely administered to patients with ovarian cancer. However, preclinical data implicate glucocorticoids in suppressing chemotherapy-mediated apoptosis in epithelial tumors. The anti-apoptotic mechanisms underlying this increased survival have been shown to require up-regulation of prosurvival genes, including serum and glucocorticoid-regulated kinase 1 (SGK1) and map kinase phosphatase 1 (MKP1)/dual specificity phosphatase 1 (DUSP1). Despite abundant preclinical data, there are no correlative studies in patients. We therefore evaluated anti-apoptotic gene expression in tumor samples from patients randomized to dexamethasone or normal saline.

Experimental design: Eighteen patients were randomized before exploratory laparotomy for suspected ovarian cancer. Dexamethasone or normal saline was administered i.v. following anesthesia. Ovarian and omental tumor samples were collected intra-operatively before and after infusion. Samples were analyzed for histology and glucocorticoid receptor expression by immunohistochemistry. SGK1 and MKP1/DUSP1 mRNA levels were determined using quantitative real-time PCR.

Results: Ten patients were evaluable. At 30 min postinfusion, tumor samples from five patients receiving dexamethasone revealed an average SGK1 mRNA induction of 6.1-fold (SEM, +/-2.6) compared with only 1.5-fold (SEM, +/-0.4) in tumor samples from five patients receiving normal saline (P = 0.028). Average MKP1/DUSP1 mRNA expression was increased by 8.2-fold (SEM, +/-2.9) following dexamethasone versus 1.1-fold (SEM, +/-0.4) following normal saline (P = 0.009). All samples expressed glucocorticoid receptor.

Conclusion: Glucocorticoid administration to patients is associated with rapid up-regulation of SGK1 and MKP1 expression in ovarian tumors. This finding supports the hypothesis that pharmacologic doses of glucocorticoids may decrease chemotherapy effectiveness in ovarian cancer patients through increased anti-apoptotic gene expression.

Conflict of interest statement

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Figures

Fig. 1

Dexamethasone inhibits paclitaxel-induced apoptosis in ovarian cancer cells. A, Western blot analysis of the glucocorticoid receptor isoforms and β-actin was done using cell lysates from the ovarian cancer cell lines SKOV3 and HeyA8. B, SKOV3 cells were cultured in the absence of growth factors for 24 h and then treated with dexamethasone (10−6 mol/L) or ethanol for 1 h before paclitaxel at varying concentration of paclitaxel (10−6, 10−7, 10−8 mol/L). Apoptosis assays were done 30 h following chemotherapy treatment using 4′,6-diamidino-2-phenylindole staining and fluorescent microscopy to score the percentage of apoptotic cells. Experiments were done in triplicate. Bars, ±SE (SEM).

Fig. 2

SGK1 and MKP1/DUSP1 mRNA and protein analysis following dexamethasone in SKOV3 cells. A, ovarian cancer cells SKOV3 were treated with vehicle (ethanol), dexamethasone (10−6 mol/L) or dexamethasone/RU486 (10−7 mol/L), quantitative real-time PCR analysis of SGK1 mRNA was done at 1 and 4 h after treatment, and expression levels were normalized to GAPDH mRNA. Average expression levels of SGK1 mRNA in the cells treated with vehicle, dexamethasone, or dexamethasone/RU486 are shown along with SD. **, significant increase in SGK1 mRNA at 4 h in dexamethasone compared with vehicle-treated cells (P < 0.05). B, Western blot analysis of the expression of SGK1 isoforms and β-actin in SKOV3 cells treated with dexamethasone (10−6 mol/L) for 4, 16, and 24 h, or vehicle alone for 24 h. All cells were cultured with the proteasome inhibitor MG132 (10 μmol/L) for 4 h before harvesting. C, MKP1/DUSP1 mRNA quantitative real-time PCR analysis. **, significant increase in MKP1/DUSP1 mRNA levels at 1 h (P < 0.01) and 4 h (P < 0.05) compared with vehicle alone. D, the corresponding MKP1/DUSP1 Western blot. β-actin was used as a loading control.

Fig. 3

SGK1 and MKP1/DUSP1 mRNA and protein analysis following dexamethasone in HeyA8 ovarian cancer cells. A, HeyA8 cells were treated with vehicle (ethanol), dexamethasone (10−6 mol/L), or dexamethasone/RU486 (10−7), and quantitative real-time PCR analysis of SGK1 mRNA was done at 1 and 4 h, followed by normalization of expression levels to GAPDH mRNA. Average expression levels of SGK1 mRNA in the cells treated with vehicle, dexamethasone, or dexamethasone/RU486 along with SD. **, significant increase in SGK1 mRNA at 1 (P < 0.01) and 4 (P < 0.05) h in dexamethasone versus vehicle-treated cells. B, Western blot analysis with the expression of SGK1 and β-actin in HeyA8 cells treated with dexamethasone (10−6 mol/L) for 1, 16, and 24 h, or vehicle for 24 h. All cells were cultured with the proteasome inhibitor MG132 (10 μmol/L) for 4 h before cell harvesting. C, MKP1/DUSP1 mRNA by quantitative real-time PCR analysis. **, a significant increase in MKP1 levels at 1 h (P < 0.01) and 4 h (P < 0.05). D, corresponding MKP1/DUSP1 Western blot, with β-actin as a loading control.

Fig. 4

Human subjects’ protocol schema.

Fig. 5

Glucocorticoid receptor immunohistochemistry of representative tissue samples from enrolled patients. A, ovarian papillary serous carcinoma; B, omental metastasis of an ovarian carcinoma; C, benign serous cyst adenoma; D, primary nonimmune mouse immunoglobulin G antibody (negative control). Top and bottom, different magnifications (original magnification, ×100 and ×200, respectively).

Fig. 6

Dexamethasone administration to patients is associated with increased expression of SGK1 and MKP1/DUSP1 mRNA in tumor samples. A, quantitative real-time PCR analysis of SGK1 mRNA was done on tumor samples obtained immediately before dexamethasone 20 mg or normal saline administration and 30 min after infusion. Expression levels were normalized to GAPDH mRNA as an internal control. The average-fold change relative to baseline in dexamethasone and normal saline – treated patient tissues together with the SE (SEM; bars). **, significant increase in SGK1 mRNA levels at 30 min in patients treated with dexamethasone (P = 0.028). B, MKP1/DUSP1 mRNA expression from the same samples as in A. **, significant increase in MKP1/DUSP1 mRNA levels at 30 min in patients treated with dexamethasone compared with normal saline (P = 0.009). C, Western blot analysis of SGK1 and β-actin in tumor tissue samples obtained at 0, 0.5, and 2 h from a patient randomized to receive dexamethasone.