Niclosamide suppresses renal cell carcinoma by inhibiting Wnt/β-catenin and inducing mitochondrial dysfunctions

Abstract

Purpose: To investigate the effects of anthelminthic drug niclosamide in renal cell carcinoma (RCC) and the underlying mechanisms of its action.

Methods: The effects of niclosamide on the proliferation and apoptosis of RCC cells were examined in vitro and in vivo by using MTS, colony formation assay, flow cytometry and xenograft cancer mouse model. Mechanism studies were performed by analyzing Wnt/β-catenin signaling and mitochondrial functions in a panel of RCC cell lines.

Results: We show that niclosamide effectively targets two RCC cell lines through inhibiting proliferation and anchorage-independent colony formation, and inducing apoptosis. It also enhances the inhibitory effects of chemotherapeutic drug cisplatin in two independent in vivo RCC xenograft mouse models. Mechanistically, niclosamide decreases β-catenin levels and therefore suppresses Wnt/β-catenin activities. Overexpression of β-catenin partially reverses the inhibitory effects of niclosamide in RCC cells, demonstrating that besides β-catenin, other mechanisms are involved in niclosamide's anti-cancer activity. Indeed, we further show that niclosamide induces mitochondrial dysfunctions as shown by the decreased level of mitochondrial membrane potential and respiration, resulting in decreased ATP levels and increased reactive oxygen species (ROS) levels.

Conclusions: Our findings support the inhibitory effects of niclosamide in cancer and provide better understanding on its underlying mechanism. Our data suggests that niclosamide is a useful addition to the treatment armamentarium for RCC.

Keywords: Mitochondria; Niclosamide; Renal cell carcinoma; Wnt/β-catenin.

Fig. 1

Niclosamide inhibits proliferation and anchorage-independent colony formation and induces apoptosis in RCC cell lines. a Niclosamide decreases proliferation of A-498 and SW-839 cells. b Niclosamide induces apoptosis of A-498 and SW-839. c Representative images taken at 14 days of an anchorage-independent colony formation assay. d Quantification of colonies show the dose-dependent inhibitory effect of niclosamide on colony formation in RCC cells. *p < 0.05, compared to control

Fig. 2

Niclosamide inhibits Wnt/β-catenin signaling in RCC cells. a Niclosamide decreases intracellular β-catenin levels in A-498 and SW-839 cells. Cells were treated with niclosamide for 24 h prior to WB analysis. b Niclosamide inhibits TOPflash activation. RCC cells transfected with TOPflash plasmid were treated with niclosamide as indicated. RLU, relative light units. c β-Catenin level is increased n A-498 and SW-839 cells transfected with β-catenin overexpression plasmid. Overexpression of β-catenin partially rescued the effects of niclosamide in inhibiting proliferation (d) and colony formation (e) and inducing apoptosis (f) in RCC cells. *p < 0.05, compared to control or p-Vector

Fig. 3

Niclosamide impairs mitochondrial function and induces oxidative stress. Niclosamide decreases mitochondrial membrane potential (a), inhibits mitochondrial respiration (b), increases ROS levels (c) and reduces ATP levels (d) in RCC cells. *p < 0.05, compared to control

Fig. 4

Niclosamide inhibits RCC tumor growth and enhances the inhibitory effect of cisplatin in vivo. Niclosamide inhibited growth of RCC tumors derived from A-498 (a) and SW-839 (b) cells as a single drug. Combination of niclosamide and cisplatin significantly inhibited much more tumor growth throughout the duration of treatment in two xenograft RCC models. *p < 0.05, compared to control or single arm