Drug repositioning of mevalonate pathway inhibitors as antitumor agents for ovarian cancer

Abstract

Drug repositioning is an alternative strategy redirecting existing drugs for new disease. We have previously reported an antitumor effect of statins, antidyslipidemic drugs, on ovarian cancer in vitro and in vivo. In this study, we investigated the antitumor effects of other mevalonate pathway inhibitors and the mechanism of the antitumor effect from a metabolic perspective. The effects of inhibitors of the mevalonate pathway on tumor cell growth were evaluated in vitro. Bisphosphonates that inhibit this pathway are commonly used as antiosteoporotic drugs, and antitumor effects of the bisphosphonate were examined in vitro and in vivo. Metabolites in SKOV3 ovarian cancer cells were analyzed before and after lovastatin treatment, using capillary electrophoresis-mass spectrometry. All mevalonate pathway inhibitors showed concentration-dependent inhibitory effects on tumor cell growth. Particularly marked effects were obtained with inhibitors of farnesyltransferase and geranylgeranyltransferase. The bisphosphonate was also shown to have an antitumor effect in vivo. The expression of autophagy marker LC3A/3B was increased in cells after treatment. In metabolomics analysis, lovastatin treatment increased the metabolites involved in the tricarboxylic acid cycle while reducing the metabolites associated with glycolysis. Also it decreased glutathione and resulted to work with chemotherapeutic agents synergistically. Inhibition at any point in the mevalonate pathway, and especially of farnesyl pyrophosphate and geranylgeranyl pyrophosphate, suppresses growth of ovarian cancer cells. Inhibition of this pathway may induce autophagy, cause a shift to activation of the tricarboxylic acid cycle and enhance susceptibility to chemotherapy. Drug repositioning targeting mevalonate pathway for ovarian cancer deserves consideration for clinical application.

Keywords: bisphosphonate; drug repositioning; mevalonate pathway; ovarian cancer; statin.

Figure 1. Selected schema of the mevalonate pathway with inhibitors used in this study

Figure 2. A representative inhibitors on the mevalonate pathway significantly inhibited cell proliferation of ovarian cancer and induced autophagy

(A) 6-Fluoromevalonate, inhibitor for mevalonate-pyrophosphate decarboxylase, significantly inhibited the cell proliferation and induced the expression of autophagy marker. (B) YM-53601, inhibitor for squalene synthase, significantly inhibited the cell proliferation and induced the expression of autophagy marker. (C) Lonafarnib, inhibitor for farnesyl transferase, significantly inhibited the cell proliferation and induced the expression of autophagy marker. (D) GGTI-298, inhibitor for geranylgeranyl transferase, significantly inhibited the cell proliferation and induced the expression of autophagy marker. Viable cells were measured at 24-hour intervals over a 120-hour period. Data are presented as the mean ±SD (n = 3).

Figure 3. Bisphosphonate-mediated anti-tumor effect in vitro and in vivo

(A) IC50 with alendronate significantly inhibited the ovarian cancer cell proliferation and induced the expression of autophagy marker. (B) Alendronate administration leads to a significant decrease in ovarian mass in mogp-TAg mice as compared to controls (**p < 0.01). (C) Representative images of Hematoxylin-Eosin, Ki-67 and LAMC1 staining on tissue sections from fallopian tubes of mogp-TAg mice. (D) Summary of Ki-67 and LAMC1 staining results. Bar graphs depict the percentage of Ki-67–positive or LAMC1–positive epithelial cells among total fallopian tube epithelial cells per section. In each experimental group, data were collected from 10 representative sections from each mouse. **p < 0.01. (E) Body weight and serum levels of cholesterol and triglyceride in mogp-TAg mice models. Oral administration of 15 mg/Kg alendronate in the mogp-TAg mice reduced serum levels of cholesterol and triglyceride (**p < 0.01), but did not affect body weight at this dose.

Figure 4. Lovastatin administration increased TCA cycle related metabolites and interfered glycolysis

(A) Principal component (PC) analysis was conducted in order to compare the overall metabolomic profiles in harvested cells. 24C; control at 24 hours, 24S; lovastatin at 24 hours, 48C; control at 48 hours, 48S; lovastatin at 48 hours. (B) Increased metabolites, Tryptophan, Phenylalanine, Valine and Methionine, in lovastatin treated cells (*p < 0.05, **p < 0.01). (C) The ratio of NADH/NAD+ was calculated in lovastatin treated cells compared to control cells. It must be decreased when TCA cycle is activated (*p < 0.05). (D) The level of acetyl-CoA and lactate in lovastatin treated cells compared to control cells (*p < 0.05). (E) Graphs of increased metabolites by lovastatin treatment in TCA cycle.

Figure 5. Lovastatin decrease Glutathione, and resulted to work with chemotherapeutic agents synergistically

(A) The serum value of total, reduced and oxidized glutathione in lovastatin mediated cells (*p < 0.05). (B) Combination index was calculated in lovastatin treatment with paclitaxel or carboplatin.