Ivermectin induces apoptosis of esophageal squamous cell carcinoma via mitochondrial pathway

Abstract

Background: Esophageal squamous cell carcinoma (ESCC) is the most predominant primary malignant tumor among worldwide, especially in China. To date, the successful treatment remains a mainly clinical challenge, it is imperative to develop successful therapeutic agents.

Methods: The anti-proliferative effect of ivermectin on ESCC is investigated in cell model and in nude mice model. Cell apoptosis was assessed using flow cytometry, TUNEL assay and western blotting. Mitochondrial dysfunction was determined by reactive oxygen species accumulation, mitochondrial membrane potential and ATP levels.

Results: Our results determined that ivermectin significantly inhibited the proliferation of ESCC cells in vitro and in vivo. Furthermore, we found that ivermectin markedly mediated mitochondrial dysfunction and induced apoptosis of ESCC cells, which indicated the anti-proliferative effect of ivermectin on ESCC cells was implicated in mitochondrial apoptotic pathway. Mechanistically, ivermectin significantly triggered ROS accumulation and inhibited the activation of NF-κB signaling pathway and increased the ratio of Bax/Bcl-2.

Conclusions: These finding indicated that ivermectin has significant anti-tumour potential for ESSC and may be a potential therapeutic candidate against ESCC.

Keywords: Apoptosis; ESCC; Ivermectin; Mitochondrial; NF-κB.

Fig. 1

Ivermectin inhibits proliferation of ESCC cells in vitro. A. Viability of cells was assessed by MTT assay. B. The morphological changes of ESCC cells were observed by phase contrast microscopy at 200× magnification, Scale bar = 50 μm; The anti-proliferative effect of ivermectin on ESCC cells was determined by LDH assay (C); colony formation (D), Scale bar = 10 mm; and EdU incorporation assays (E), Scale bar = 50 μm; F. Cell cycle of ESCC cells after treatment with was performed by flow cytometry. Values represent mean ± S.E.M. of 3 independent experiments. ^*P < 0.05, ^**P < 0.01; ^***P < 0.001.

Fig. 2

Ivermectin promotes apoptosis of ESCC cells. Western blotting (A) and qRT-PCR (B) analysis for the expression levels of Bax and Bcl-2 after ivermectin treatment; C. The expression of caspase cascade (cleaved-caspase 9, cleaved-caspase 3, PARP, cleaved PARP) were analyzed by western blotting; D. Cells were observed by fluorescence microscopy after staining with Hoechst 33342, Scale bar = 100 μm; E. Ivermectin-induced ESCC cells apoptosis was determined by TUNEL assay (200×), Scale bar = 50 μm; F. Ivermectin-induced ESCC cells apoptosis was determined by PI/Annexin-V assay using flow cytometry. ^*P < 0.05, ^**P < 0.01; ^***P < 0.001.

Fig. 3

Ivermectin mediates mitochondrial dysfunction of ESCC cells. A. Intracellular ROS levels were investigated by DCFH-DA fluorescence in ESCC cells treatment with indicated; B. The expression levels of Bax and Bcl-2 were determined by western blot analysis; C. The expression of caspase cascade (cleaved-caspase 9, cleaved-caspase 3, PARP, cleaved PARP) were analyzed by western blotting; D. Mitochondrial membrane potential was observed by fluorescence microscope at 200 × magnifications, Scale bar = 50 μm; E. ATP production of ESCC cells after treated with ivermectin was detected; F. The mitochondrial DNA copy number in ESCC cells treatment with invermectin was evaluated using qRT-PCR. ^*P < 0.05, ^**P < 0.01.

Fig. 4

Ivermectin induces apoptosis of ESCC cells through NF-κB pathway. A and C. Western blotting analysis for the expression levels of p65, p-p65, p-IκBα and IκBα in ESCC cells after ivermectin treatment; B and D. Quantified for the protein expression levels of (A) and (C). ^*P < 0.05, ^**P < 0.01; ^***P < 0.001.

Fig. 5

Ivermectin suppressed xenograft growth of ESCC cells in vivo. A, B and C. Tumor growth curves and weight of subcutaneous xenograft tumor model developed from ESCC cells treatment with ivermectin as indicated (n=5); D and E. Representative immunohistochemistry images of Ki67, ROS1, p-p65 and cleaved-caspase 3 in xenograft tumor developed from KYSE-70 and KYSE-30 cells treatment with ivermectin as indicated. Scale bar: 300μm. ^*P < 0.05, ^**P < 0.01; ^***P < 0.001.

Fig. 6

Schematic depicting the anti-proliferative potential of ivermectin on ESCC cells.