Anticancer Activity of Metformin, an Antidiabetic Drug, Against Ovarian Cancer Cells Involves Inhibition of Cysteine-Rich 61 (Cyr61)/Akt/Mammalian Target of Rapamycin (mTOR) Signaling Pathway

Abstract

BACKGROUND Ovarian cancer is considered one of the lethal cancers responsible for high mortality and morbidity across the world. The prognosis and the survival rate of ovarian cancer is far from decent. Cysteine-rich 61 (Cyr61) also known as CCN1, is a member of CCN-family of growth factors, reported to be significantly overexpressed in several malignancies which include, but are not limited to, ovarian cancer. Recent studies have revealed that women with type 2 diabetes mellitus have an elevated risk of ovarian cancer. Hence, administration of an antidiabetic drug with anticancer effects such as metformin may act as an effective therapeutic regime against ovarian cancer. MATERIAL AND METHODS Cell viability and apoptosis were examined by MTT and Annexin V/PI double staining respectively. Cell migration was determined by Boyden Chamber assay. Transient knockdown of Cyr61 in ovarian cancer cells was achieved by transecting the cells with siRNA for Cyr61using Lipofectamine 2000. RESULTS Our results indicated that treatment of ovarian cancer cells with metformin caused significant downregulation of Cyr61 protein expression levels ultimately favoring apoptosis. Transient knockdown of Cyr61 resulted in the inhibition of cell proliferation and migration. This was also associated with the concomitant downregulation of pAkt and pmTOR confirming the role of Cyr61 as an upstream modulator of Akt signaling. Conversely the extracellular supplementation of recombinant Cyr61 attenuates the cytotoxic properties of metformin in ovarian cancer cells. CONCLUSIONS Taken together, we concluded that metformin exhibits anticancer effects and Cyr61 acts as a direct target for metformin in ovarian cancer cells.

Figure 1

Metformin induces apoptosis in ovarian cancer cells. (A) Cell viability of OVCAR-3 cells in the presence of metformin for 48 hours as determined by MTT assay. (B) Apoptosis assay for OVCAR-3 cells treated with metformin as determined by Annexin V/PI double staining. (C) Modulation of pro- and anti-apoptotic markers. The results are expressed as mean ± standard deviation of 3 independent experiments. The values were considered significant at * P<0.05.

Figure 2

Metformin inhibits colony formation in ovarian cancer cells. (A) Photographs of viable colonies of OVCAR-3 cells in the presence or absence of metformin, stained with crystal violet; (B) % colony forming unit (CFU), determined spectrophotometrically. The results are expressed as mean ± standard deviation of 3 independent experiments. The values were considered significant at * P<0.05.

Figure 3

Metformin inhibits invasiveness of ovarian cancer cells. (A) Invasion was determined by the Boyden Chamber assay. (B) Extent of invasion as determined spectrophotometrically. The results are expressed as mean ± standard deviation of 3 independent experiments. The values were considered significant at * P<0.05.

Figure 4

Effect of Cyr61 on oncogenic potential and PI3K/Akt/mTOR axis in ovarian cancer cells. (A) Transient knockdown of Cyr61 by siRNA method in OVCAR-3 cells. (B) qRT-PCR analysis showing the expression of Cyr61 in Cyr61SiRNA transfected OVCAR-3 cells. (C) Viability of Cyr61 siRNA and scrambled siRNA transfected OVCAR-3 cells. (D) Boyden Chamber invasion assay of Cyr61 siRNA and scrambled siRNA transfected OVCAR-3 cells. (E) PI3K/Akt/mTOR axis in OVCAR-3 cells. All the experiments were carried out in triplicates. The values were considered significant at * P<0.05.

Figure 5

Effect of metformin on the PI3K/Akt/mTOR pathway. Cultured OVCAR-3 cells were treated with different doses of metformin (0–20 mM) for 48 hours and western blots for p-Akt (Ser 473), Akt, p-mTOR (Ser 2448) and mTOR were performed. (A) Western blot analysis for PI3K (p85), PI3K, p-Akt ^{Ser 473}, Akt, p-mTOR ^{Ser 2448}, mTOR, and β-actin used as a loading control. Relative band intensities for (B) p-PI3K (p85), (C) p-Akt ^{Ser 473}, and (D) p-mTor ^{Ser 2448}. The results are expressed as mean ± standard deviation of 3 independent experiments. The values were considered significant at * P<0.05.

Figure 6

Cyr61 regulates metformin action in ovarian cancer cells (A) and (B) the effect of metformin on Cyr61 expression in OVCAR-3 cells. (C) Viability of metformin-treated OVCAR-3 cells in the presence of human recombinant Cyr61. All the experiments were carried out in triplicates. The values were considered significant at * P<0.05.

Figure 7

Cyr61 regulates chemoresistance properties of ovarian cancer cells. (A) Cell viability of Cyr61 siRNA and scrambled siRNA transfected OVCAR-3 cells in the presence of paclitaxel. (B) Combination of paclitaxel and metformin.