Tigecycline as a dual inhibitor of retinoblastoma and angiogenesis via inducing mitochondrial dysfunctions and oxidative damage

Abstract

Retinoblastoma is the most common intraocular malignancy in children with poor prognosis. The progression of retinoblastoma is dependent on a robust angiogenic response. Targeting both retinoblastoma cells and angiogenesis may therefore provide an alternative therapeutic strategy in retinoblastoma. Here, we demonstrated the inhibitory effects of tigecycline, a FDA-approved antibiotic, in retinoblastoma and angiogenesis in vitro and in vivo. We showed that tigecycline significantly inhibited growth and induced caspase-dependent apoptosis of multiple retinoblastoma cell lines. Tigecycline also effectively inhibited angiogenesis through suppressing capillary network formation, migration, proliferation and survival of human retinal microvascular endothelial cell (HREC). Mechanistically, tigecycline acts on both retinoblastoma cells and HREC via inhibiting mitochondrial protein translation, resulting in mitochondrial dysfunction, energy crisis, and oxidative damage. Importantly, we demonstrated the in vivo efficacy of tigecycline in inhibiting retinoblastoma and angiogenesis, and inducing oxidative stress on xenograft mouse model. In addition, ATP levels and growth rates were largely affected in retinoblastoma ρ0 cells that lacked mitochondrial respiration. Our work provides systematic pre-clinical evidence for repurposing tigecycline from its traditional use for retinoblastoma treatment. Our work demonstrates the essential roles of mitochondrial metabolism in both retinoblastoma and its angiogenesis.

Figure 1

Tigecycline significantly inhibits proliferation and induces caspase-dependent apoptosis in retinoblastoma cells. Tigecycline at 5, 10 and 20 µM inhibits proliferation (A) and induces apoptosis (B) of retinoblastoma cell lines: Y79, RB116 and WERI-Rb1. (C) Tigecycline at 20 µM significantly induces apoptosis of retinoblastoma cells in a time-dependent manner. (D) Tigecycline (20 µM) is ineffective in inducing apoptosis in the presence of a pan-caspase inhibitor Z-VAD-fmk (50 µM). *p < 0.05, compared to control.

Figure 2

Tigecycline inhibits retinal angiogenesis. (A) Representative images of in vitro capillary network formation showing dose-dependent inhibitory effect of tigecycline on retinal angiogenesis. In vitro capillary tube formation was fully formed at 6 hours after plating primary human retinal microvascular endothelial cells (HREC) onto complete Matrigel. Tigecycline significantly decreases migration (B), proliferation (C) and increases apoptosis (D) of HRECs. *p < 0.05, compared to control.

Figure 3

Tigecycline inhibits mitochondrial translation in retinoblastoma and HREC. Tigecycline decreases protein level (A) and increases mRNA level (B) of Cox-1 and -2 without affecting protein and mRNA levels of Cox-4 in retinoblastoma cells and HREC. Transcript levels of each gene was normalized with β-actin. (C) Tigecycline suppresses mitochondrial respiratory complex I and IV but not II activity in Y79 and HREC cells. *p < 0.05, compared to control.

Figure 4

Tigecycline inhibits mitochondrial respiration and ATP production, and induces oxidative stress in retinoblastoma and HREC. Tigecycline significantly decreases basal (A) and maximal (B) OCR in a dose-dependent manner in Y79 and HREC. Tigecycline significantly decreases ATP (C) and increases mitochondrial superoxidase (D) level in Y79 and HREC. *p < 0.05, compared to control.

Figure 5

Tigecycline causes oxidative damage in retinoblastoma and HREC. Tigecycline significantly increases 8-OHdG (A) and carbonylation (B) but not MDA (C) level in Y79 and HREC. *p < 0.05, compared to control.

Figure 6

Tigecycline induces retinoblastoma cell apoptosis through its suppression of mitochondrial respiration. (A) Remarkable reduction on the mRNA expression of two mitochondrial genome-encoded (MT-ND6 and MT-CO2) but not nuclear genome-encoded (SDHA) respiratory chain enzyme subunits in Y79 ρ0. Remarkable reduction on the basal (B) and maximal (C) OCR level in Y79 ρ0 cells. Significant reduction in ATP levels (D) and minimal growth rate (E) in Y79 ρ0 cells. (E) Tigecycline (20 µM) is ineffective in inducing apoptosis in Y79 ρ0 cells. *p < 0.05, compared to Y79 wildtype.

Figure 7

Tigecycline significantly inhibits retinoblastoma growth in mice without toxicity. (A) No significant changes on mouse body weight in control and tigecycline-treated groups. (B) Tigecycline significantly inhibits retinoblastoma growth in vivo. *p < 0.05, compared to control. ns, not significant.

Figure 8

Tigecycline significantly inhibits retinoblastoma angiogenesis and induces oxidative stress in vivo. (A) Representative photos of immunohistochemistry of microvascular marker CD31, oxidative stress markers HEL and 4-HNE staining. Tigecycline significantly inhibits retinoblastoma angiogenesis (B) and increases oxidative stress (C,D) in vivo. Intraperitoneal tigecycline at 60 mg/kg once per day were given to the mice. *p < 0.05, compared to control.