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. 2016 Jan 5;7(1):946-60.
doi: 10.18632/oncotarget.5834.

The HMG-CoA reductase inhibitor, simvastatin, exhibits anti-metastatic and anti-tumorigenic effects in ovarian cancer

Jessica E Stine  1 , Hui Guo  1   2 , Xiugui Sheng  2 , Xiaoyun Han  1   2 , Monica N Schointuch  1 , Timothy P Gilliam  1 , Paola A Gehrig  1   3 , Chunxiao Zhou  1   3 , Victoria L Bae-Jump  1   3
Affiliations

The HMG-CoA reductase inhibitor, simvastatin, exhibits anti-metastatic and anti-tumorigenic effects in ovarian cancer

Jessica E Stine et al. Oncotarget. .

Abstract

Ovarian cancer is the 5th leading cause of cancer death among women in the United States. The mevalonate pathway is thought to be a potential oncogenic pathway in the pathogenesis of ovarian cancer. Simvastatin, a 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) inhibitor, is a widely used drug for inhibiting the synthesis of cholesterol and may also have anti-tumorigenic activity. Our goal was to evaluate the effects of simvastatin on ovarian cancer cell lines, primary cultures of ovarian cancer cells and in an orthotopic ovarian cancer mouse model. Simvastatin significantly inhibited cellular proliferation, induced cell cycle G1 arrest and apoptosis, and caused cellular stress via reduction in the enzymatic activity of HMGCR and inhibition of the MAPK and mTOR pathways in ovarian cancer cells. Furthermore, simvastatin induced DNA damage and reduced cell adhesion and invasion. Simvastatin also exerted anti-proliferative effects on primary cell cultures of ovarian cancer. Treatment with simvastatin in an orthotopic mouse model reduced ovarian tumor growth, coincident with decreased Ki-67, HMGCR, phosphorylated-Akt and phosphorylated-p42/44 protein expression. Our findings demonstrate that simvastatin may have therapeutic benefit for ovarian cancer treatment and be worthy of further exploration in clinical trials.

Keywords: HMGCR; apoptosis; invasion; ovarian cancer; simvastatin.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors have no potential conflicts of interest to report.

Figures

Figure 1
Figure 1. Simvastatin inhibited the growth of ovarian cancer cells and HMGCR activity
Hey and SKOV3 cells were cultured for 24 h and then treated with varying concentrations of simvastatin in 96 well plates for 72 h. Cell proliferation was assessed by MTT assay A. The effect of simvastatin on its target, HMGCR, was examined by Western blot analysis. Simvastatin treatment resulted in a dose-dependent decrease in expression of HMGCR protein in both cell lines B. HMGCR activity in ovarian cancer cells was measured via HMGCR Assay. Treatment with simvastatin for 24 h resulted in a dose-dependent decrease in HMGCR activity in both the Hey and SKOV3 cell lines C. Each experiment was performed three times. C in graphs refers to control. (*p < 0.05, **p < 0.01).
Figure 2
Figure 2. Simvastatin induced cell cycle G1 arrest in ovarian cancer cells
The Hey A. and SKOV3 B. cell lines were treated with the indicated doses of simvastatin (1–25 uM) for 24 h. Cell cycle analysis was performed by Cellometer. Simvastatin markedly induced cell cycle G1 arrest in both cell lines in a dose dependent manner. Each experiment was performed three times. C in graphs refers to control. (*p < 0.05, **p < 0.01).
Figure 3
Figure 3. Simvastatin increased apoptosis in ovarian cancer cells
The Hey A. and SKOV3 B. cell lines were cultured for 24 h and then treated with simvastatin at different doses for 24 h. Apoptosis was examined by Annexin V assay in Cellometer. Caspase-3, Caspase-9 and BCL-2 were determined by Western immunoblotting after exposure to simvastatin for 10 h or 24 h C and D. Each experiment was performed three times. C in graphs refers to control. (*p < 0.05, **p < 0.01).
Figure 4
Figure 4. Simvastatin caused cellular stress and DNA damage in ovarian cancer cells
The Hey A. and SKOV3 B. cell lines were treated with simvastatin at different concentrations for 18 h and reactive oxygen species (ROS) level was determined using DCFH-DA dye on a plate reader. PERK and Bip were determined by Western immunoblotting after exposure to simvastatin for 24 h C. DNA damage in the Hey D. and SKOV3 E. cell lines was analyzed by QPCR assay after treatment with simvastatin for 24 h. Each experiment was performed three times. (*p < 0.05, **p < 0.01). C in graphs refers to control.
Figure 5
Figure 5. Simvastatin reduced on adhesion and invasion in ovarian cancer cells
The Hey A, C. and SKOV3 B, D. cell lines were cultured for 24 h and then treated as indicated with simvastatin in a laminin coated 96 well plate for 2 h to assess adhesion or a BME coated 96 transwell plate for 24 h to assess invasion, respectively. The data represents relative inhibition in each cell line. VEGF was measured by ELISA assay in culture media E. and cell lysates F. after a 36 h exposure to simvastatin. Each experiment was performed three times. (*p < 0.05, **p < 0.01). C in graphs refers to control.
Figure 6
Figure 6. Simvastatin inhibited MAPK and AKT/mTOR pathways in ovarian cancer cells
Hey and SKOV3 cells were treated with simvastatin at different doses for 24 h. Phosphorylated-p42/44, phosphorylated-AKT and phosphorylated-S6 were assessed by Western blotting. Simvastatin inhibited the activity of MAPK and AKT/mTOR pathways in Hey and SKOV3 cells. Each experiment was performed two times. (*p < 0.05, **p < 0.01).
Figure 7
Figure 7. Simvastatin reduced tumor growth of orthotropic xenografts of serous ovarian cancer
M909 cells were injected into left side of the ovarian bursa of 6–8 week old mice. When the tumors reached ~50 mm3 (approximately 12 days after injection), the mice were treated with placebo or 3 mg/kg simvastatin once a day for 4 weeks. Tumor volume A. and weight B. were recorded during or after 4 weeks treatment. Simvastatin reduced serum cholesterol in the mice C. VEGF was measured by ELISA assay in mouse serum and tumor tissues D. The changes of Ki-67, cleaved caspase 3, HMGCR, phosphorylated-AKT and phosphorylated-p42/44 were assessed by immunohistochemistry in ovarian tumor tissues E. (*p < 0.05. **p < 0.01).
Figure 8
Figure 8. Simvastatin decreased cell proliferation in primary cultures of ovarian cancer cells
Cell proliferation was assayed by MTT assay in seven primary cultures of ovarian cancer cells after 72 h treatment with simvastatin A. The IC50 value for primary cultures is shown in B. HMGCR protein expression was detected by western blotting in the seven untreated primary cultures of ovarian cancer cells C, D. HMGCR expression did not predict sensitivity to simvastatin in ovarian cancer cells.
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