Korean J Physiol Pharmacol.  2024 May;28(3):197-207. 10.4196/kjpp.2024.28.3.197.

Tivozanib-induced activation of the mitochondrial apoptotic pathway and suppression of epithelial-to-mesenchymal transition in oral squamous cell carcinoma

• Choi NE
• Park SC
• Kim IR

• KMID: 2555662
• DOI: http://doi.org/10.4196/kjpp.2024.28.3.197

Abstract

The potential of tivozanib as a treatment for oral squamous cell carcinoma (OSCC) was explored in this study. We investigated the effects of tivozanib on OSCC using the Ca9-22 and CAL27 cell lines. OSCC is a highly prevalent cancer type with a significant risk of lymphatic metastasis and recurrence, which necessitates the development of innovative treatment approaches. Tivozanib, a vascular endothelial growth factor receptor inhibitor, has shown efficacy in inhibiting neovascularization in various cancer types but has not been thoroughly studied in OSCC. Our comprehensive assessment revealed that tivozanib effectively inhibited OSCC cells. This was accompanied by the suppression of Bcl-2, a reduction in matrix metalloproteinase levels, and the induction of intrinsic pathway-mediated apoptosis. Furthermore, tivozanib contributed to epithelial-to-mesenchymal transition (EMT) inhibition by increasing E-cadherin levels while decreasing N-cadherin levels. These findings highlight the substantial anticancer potential of tivozanib in OSCC and thus its promise as a therapeutic option. Beyond reducing cell viability and inducing apoptosis, the capacity of tivozanib to inhibit EMT and modulate key proteins presents the possibility of a paradigm shift in OSCC treatment.

Keyword

Apoptosis; EMT suppression; Squamous cell carcinoma of head and neck; Tivozanib

Figure

• 

  Fig. 1 Tivozanib exhibits cytotoxic and antiproliferative effects and induces cell cycle arrest in OSCC cells. (A) Ca9-22 and CAL27 cells were treated with varying concentrations of tivozanib (0–200 μM) for 24 h. (B, C) Cells were treated with 1 or 10 μM tivozanib for 7 days. Cell proliferation was assessed by staining with 1% crystal violet. Cell viability and proliferation values are presented as percentages (compared to control cells with 0 μM tivozanib). (D) Cells were treated with 0–75 μM tivozanib for 24 h. After staining with PI, the cells were analyzed using FACS. The proportion of cells in the G2 phase after tivozanib treatment is presented as a histogram. (E) The protein levels of cell cycle-related factors were confirmed via Western blot. β-actin was used as a loading control. The results are presented as mean ± SD and were derived from three independent experiments (Ca9-22: *p < 0.05, **p < 0.01, ***p < 0.001; CAL27: #p < 0.05, ##p < 0.01, ###p < 0.001). OSCC, oral squamous cell carcinoma; PI, propidium iodide.

• 

  Fig. 2 Apoptotic responses in OSCC cells induced by tivozanib. After treating cells with 0–75 μM tivozanib for 24 h, (A) nuclear condensation and formation were observed. The number of condensed nuclei was quantified and is displayed in the graph. (B) Apoptosis in OSCC cells following tivozanib treatment was quantified using annexin V/PI staining and FACS. (C) The levels of apoptosis-related proteins were analyzed via Western blot. β-actin was used as a loading control. The results are presented as mean ± SD and were derived from three independent experiments (Ca9-22: *p < 0.05, **p < 0.01; CAL27: #p < 0.05, ##p < 0.01). OSCC, oral squamous cell carcinoma; PI, propidium iodide.

• 

  Fig. 3 Intrinsic pathway-mediated apoptosis in OSCC cells triggered by tivozanib. Cells were treated with 0.75 μM tivozanib for 24 h. After staining with JC-1, the cells were observed using fluorescence microscopy (A) and FACS (B). The green fluorescence signal in the cells treated with tivozanib was compared with that in the control cells. (C) An increase in the release of cytochrome c from the mitochondria suggested the activation of apoptotic pathways. (D) The levels of proteins involved in intrinsic pathway-mediated apoptosis were analyzed via Western blot. β-actin was used as a loading control. OSCC, oral squamous cell carcinoma; AIF, apoptosis-inducing factor.

• 

  Fig. 4 Tivozanib inhibited EMT in OSCC cells. (A, B) The wound area and number of invaded cells both significantly decreased with tivozanib treatment. (C) Tivozanib regulated the expression of EMT-related proteins in OSCC cells. (D) qPCR analysis of the mRNA levels of EMT-related markers. (E) E-cadherin expression increased with tivozanib treatment, as confirmed by confocal microscopy. The results are expressed as mean ± SD. Data were derived from three independent experiments (Ca9-22: *p < 0.05, **p < 0.01; CAL27: #p < 0.05). EMT, epithelial-to-mesenchymal transition; OSCC, oral squamous cell carcinoma; MMP9, matrix metalloproteinase 9; VEGFR2, vascular endothelial growth factor receptor 2.

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