Repurposing old carbon monoxide-releasing molecules towards the anti-angiogenic therapy of triple-negative breast cancer

Abstract

Triple-negative breast cancer (TNBC) is defined by the lack of expression of the oestrogen and progesterone receptors and HER-2. Recently, carbon monoxide (CO) was found to behave as an important endogenous signalling molecule and to suppress VEGF receptor-2 (VEGFR-2) and protein kinase B phosphorylation. Given that anti-angiogenic drugs exist as one of the few available targeted therapies against TNBC, the aim of this project was to study the effects of CO-releasing molecules (CORMs) on TNBC cell lines and the associated endothelial cells and characterise their anti-angiogenic properties that can be used for the reduction of cancer-driven angiogenesis. Four commercially available CORMs were screened for their cytotoxicity, their effects on cell metabolism, migration, VEGF expression, tube formation and VEGFR-2 activation. The most important result was the reduction in VEGF levels expressed by CORM-treated TNBC cells, along with the inhibition of phosphorylation of VEGFR2 and downstream proteins. The migration and tube formation ability of endothelial cells was also decreased by CORMs, justifying a potential re-purposing of old CORMs towards the anti-angiogenic therapy of TNBC. The additional favourable low cytotoxicity, reduction in the glycolysis levels and downregulation of haem oxygenase-1 in TNBC cells enhance the potential of CORMs against TNBC. In this study, CORM-2 remained the most effective CORM and we propose that CORM-2 may be pursued further as an additional agent in combination with existing anti-angiogenic therapies for a more successful targeting of malignant angiogenesis in TNBC.

Keywords: angiogenesis; anti-angiogenic therapy; breast cancer; carbon-monoxide releasing molecules (CORMs); triple-negative breast cancer (TNBC).

Figure 1. VEGF expression after CORM treatments

(A) Quantification of VEGF levels in the supernatants of treated MDA-MB-231 cells with 100 μM CORMs or vehicle or normal media for 6 h, 12 h or 24 h. (B) Quantification of VEGF levels in the supernatants of treated MDA-MB-231 cells with 250 μM CORMs or vehicle or normal media for 6 h, 12 h or 24 h. (C) Quantification of VEGF levels in the supernatants of treated MDA-MB-436 cells with 100 μM CORMs or vehicle or normal media for 6 h, 12 h or 24 h. (% percentage compared to media treated cells (control) ±SEM; n = 3, N = 3) (All data was statistically analysed against media treated cells using unpaired t-test with Welch's correction: ^*p < 0.05, ^**p < 0.01, ^***p < 0.001).

Figure 2. pY1175 levels after CORM treatments

(A) Expression levels of pY1175 of VEGFR2 in HUVEC after 15 min of CORM or vehicle pre-incubation and then stimulation with VEGF (100 ng/ml) for 5 min (SFM), as measured with the human pY1175-VEGFR2 ELISA kit. The first sample is the unstimulated control (Control). (Graph shows % compared to Control ±SEM; N = 3) (Data statistically analysed using unpaired Student's t-test with Welch's correction ^*p < 0.05, ^**p < 0.01). (B) Representative blot of the levels of pY1175 of VEGFR2 following the same protocol (N = 3).

Figure 3. Expression of VEGFR2 pathway proteins in HUVEC after CORM pre-treatments and VEGF stimulation

(A) Western blot of pFAK, pSrc and pERK1/2 following 15 min of CORM or vehicle pre-incubation and then stimulation with VEGF (100 ng/ml) for 5 min (SFM). The first sample is the unstimulated control (Control). (Blots show representative data; N = 3). (B) Quantitative assessment of pFAK, pSrc and pERK1/2 levels in HUVEC. (Graphs show % compared to Control ±SEM; N = 3) (Data statistically analysed using nonparametric (Mann-Whitney) t-test with ^*p < 0.05, ^**p < 0.01).

Figure 4. Tube formation ability of HECV after conditioned media treatments

(A) Representative images from tube formation assay with HECV cells treated with conditioned media from variable duration incubation of MDA-MB-231 cells with 100 μM CORMs or vehicle or normal media. Objective 5x, Scale bar = 132.08 μm. (B) Quantification of tube formation capacity of HECV cells after treatment with conditioned media. (% Total tube perimeter compared to vehicle ±SEM; n = 3, N = 3) (All data was statistically analysed against the conditioned media from corresponding duration vehicle treated MDA-MB-231 cells using un-paired t-test with Welch's correction: ^*p < 0.05, ^**p < 0.01, ^***p < 0.001).

Figure 5. Tube formation ability of HECV after CORM treatments

(A) Representative images from tube formation assay with HECV cells treated with 100 μM CORMs or vehicle or serum free media. Objective 5x, Scale bar = 132.08 μm. (B) Quantification of tube formation capacity of HECV cells after treatment with CORMs or vehicle or serum free media. (% total tube perimeter compared to vehicle ±SEM; n = 3, N = 3) (All data was statistically analysed against the vehicle group using un-paired t-test with Welch's correction: ^*p < 0.05, ^**p < 0.01).

Figure 6. Migratory ability of HECV after CORM treatments

(A) Representative images from a scratch wound assay in HECV cells at 0 h and 24 h after treatment with 100 μM CORMs or vehicle or normal media. Objective 5x, Scale bar = 100.37 μm. (B) Assessment of the healing, expressed as % wound confluence, at all time points tested after treatments. (% wound confluence ±SEM; n = 3, N = 3) (All data was statistically analysed against vehicle treated cells using two-way ANOVA: ^*p < 0.05, ^**p < 0.01, ^***p < 0.001). (C) Average % wound confluence at the final time point (24 h) for all treatments. (All data was statistically analysed against the vehicle group using un-paired t-test with Welch's correction: ^*p < 0.05, ^**p < 0.01, ^***p < 0.001).