Visfatin induces ovarian cancer resistance to anoikis by regulating mitochondrial activity

Abstract

Purpose: Ovarian cancer is characterized by recurrent peritoneal and distant metastasis. To survive in a non-adherent state, floating ovarian cancer spheroids develop mechanisms to resist anoikis. Moreover, ascitic fluid from ovarian cancer patients contains high levels of visfatin with anti-apoptotic properties. However, the mechanism by which visfatin induces anoikis resistance in ovarian cancer spheroids remains unknown. Here, we aimed to assess wheather visfatin which possess anti-apoptotic properties can induce resistance of anoikis in ovarian cancer spheroids.

Methods: Visfatin synthesis were examined using a commercial human visfatin ELISA Kit. Spheroid were exposed to visfatin and cell viability and caspase 3/7 activity were measured using CellTiter-Glo 3D cell viability assay and Caspase-Glo® 3/7 Assay System. mRNA and protein expression were analyzed by Real-time PCR and Western Blot analysis, respectively. Analysis of mitochondrial activity was estimated by JC-1 staining.

Results: First, our results suggested higher expression and secretion of visfatin by epithelial than by granulosa ovarian cells, and in non-cancer tissues versus cancer tissues. Interestingly, visfatin increased the proliferation/apoptosis ratio in ovarian cancer spheroids. Specifically, both the intrinsic and extrinsic pathways of anoikis were regulated by visfatin. Moreover, the effect of the visfatin inhibitor (FK866) was opposite to that of visfatin. Furthermore, both NAMPT and FK866 affected mitochondrial activity in ovarian cancer cells.

Conclusion: In conclusion, visfatin acts as an anti-apoptotic factor by regulating mitochondrial activity, leading to anoikis resistance in ovarian cancer spheroids. The finding suggest visfatin as a potential novel therapeutic target for the treatment of ovarian carcinoma with peritoneal dissemination.

Keywords: Anoikis; FK866; Mitochondrial activity; Ovarian cancer; Visfatin.

Fig. 1

Level of visfatin in ovarian surface epithelium (OSE), normal stroma, fallopian tube epithelium (FTE), tumor, tumor stroma, peritoneal tumor, FTE tumor, and metastasis (http://csiovdb.mc.ntu.edu.tw/pages/CSIOVDB_NAMPT.html)

Fig. 2

Basal levels of visfatin in non-cancer and cancer ovarian cell lines. Basal expression of mRNA encoding visfatin (NAMPT), and visfatin synthesis levels, in human ovarian non-cancer epithelial (HOSEpiC) and granulosa (HGrC1) ovarian cell lines (a, d), and in epithelial ovarian cancer cell lines (OVCAR-3) and (SKOV-3) (b, e), compared with HOSEpiC; and in ovarian granulosa tumor (KGN) cells compared with HGrC1 (c, f). Expression of NAMPT mRNA in HOSEpiC cells was set to 1.0 (RQ = relative quantity) (a, b), and expression of NAMPT mRNA in HGrC1 cells was set to RQ = 1.0. c Each bar represents the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001

Fig. 3

Effect of visfatin on the proliferation/apoptosis ratio in OVCAR-3, SKOV-3 and KGN cells. Dose-dependent effect of visfatin (10, 50, 100, 500, or 1000 ng/mL) on the proliferation to apoptosis ratio of OVCAR-3 (a), SKOV-3 (b), and KGN (c) cells. Data represent the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with control untreated cells

Fig. 4

Effect of visfatin on apoptosis of KGN and SKOV-3 cells. Caspase-3 activity after treatment with a caspase-3 inhibitor (a). Dose-dependent effect of visfatin (10, 100 and 1000 ng/mL) on caspase-3 activity in KGN (b) and SKOV-3 cells (g). Expression level of caspase -3 (c, h) and PARP1 (e, i) mRNA in KGN and SKOV-3 cells treated with visfatin (100 ng/mL) for 24 h, and levels of cleaved caspase-3 (d) and PARP1 (f) protein in KGN cells. Expression was set to RQ = 1.0 in vehicle-treated controls. Each bar represents the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001

Fig. 5

Effect of visfatin on the intrinsic and extrinsic apoptotic pathways in KGN and SKOV-3 cells. Dose-dependent effect of visfatin (10, 100, and 1000 ng/mL) on activity of caspase-8 (a, g) and caspase-9 (b, h) in KGN and SKOV-3 cells, respectively. Expression of Bax (c, d, i) and Bcl-2 (e, f, j) mRNA in KGN and SKOV-3 cells treated with visfatin (100 ng/mL) for 24 h, and levels of BID/tBID protein (k) in KGN cells. Expression of mRNA in vehicle-treated controls was set to RQ = 1.0. Each bar represents the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001

Fig. 6

Action of visfatin inhibitor (FK866) in KGN cells. Effect of the FK866 (10 and 100 nM) on cell viability (a), caspase 3/7 activity (b), and the P/A ratio (c) in KGN cells. Effect of visfatin (50 ng/mL), FK866 (10 nM), and co-treatment visfatin and FK866 on the ATP content of KGN cells (d). Each bar represents the mean ± SEM of three independent experiments. *P < 0.05 and ***P < 0.001

Fig. 7

Effect of NAMPT and FK866 on mitochodrial activity in KGN cells. Mitochondrial activity in NAMPT- (B, B’, and B”) and FK866- (D, D’, and D”) treated KGN cells. Control 1 (A, A’, and A”) represents cells incubated with medium only, while control 2 (C, C’, and C”) represents cells incubated with medium plus DMSO (0.01%). Such distinctions reflect real experimental conditions. Treatment with visfatin (NAMPT) increased red fluorescence (representing active mitochondria with a high potential) (B’); therefore, red fluorescence is predominant in the merged image (B”). Treatment with the visfatin inhibitor FK866 decreased red fluorescence (representing active mitochondria) (D’) and increased green fluorescence emitted from inactive organelles (D); therefore, green fluorescence is predominant in the merged image (D”). Scale bar: 100 μm