Serum bradykinin levels as a diagnostic marker in cervical cancer with a potential mechanism to promote VEGF expression via BDKRB2

Abstract

Bradykinin (BK) is one of the kinin peptides and preferentially binds to bradykinin B2 receptor (BDKRB2). A recent study indicated that BK played an important role in the occurrence and progression of cancer. In this study, we evaluated the serum BK levels in 130 cervical cancer (CC) cases (including 65 cases with pre‑ and post‑surgery paired samples, another 65 cases with only pre‑surgery samples), 35 cervical intraepithelial neoplasia (CIN) cases (pre‑ and post‑surgery paired) and 35 control cases. We found that BK was overexpressed in patients with CC compared to patients with CIN and the control group. When combined with squamous cell carcinoma‑related antigen (SCCA), the diagnostic efficacy of BK was prominently enhanced. Moreover, we detected the expression level of the BK receptor BDKRB2 in CC, CIN and normal cervical tissues and observed a higher expression in the CC and CIN tissues than in the normal cervix. We then explored the possible mechanisms of action of BK in promoting the progression of CC. When BK was added to the cell culture medium, human umbilical vein endothelial cell (HUVEC) angiogenesis increased and vascular endothelial growth factor (VEGF) expression in CC cell lines was also elevated. The BK antagonist, HOE140, exerted an opposite effect. The knockdown or the overexpression of BDKRB2 in CC cell lines further confirmed its oncogenic role in angiogenesis. Taken together, the findings of this study suggest that BK may be a diagnostic biomarker for CC and may notably improve the diagnostic efficacy when combined with SCCA. BK promotes the progression of CC by upregulating the expression of VEGF via BDKRB2 and subsequently facilitating angiogenesis.

Figure 1

BK is overexpressed in the serum of patients with CC. (A) The expression level of serum BK was examined in CC (n=130), CIN (n=35) and control (n=35) patients by ELISA. The average expression level of BK was significantly higher in patients with CC compared with patients with CIN (P<0.05) and control patients (P<0.01). (B) Expression level of serum BK in CC (n=65) and CIN (n=35) patients pre- and post-surgery. The pre-operative BK level was prominently higher than the corresponding post-operative BK level in patients with CC (P<0.0001). For patients with CIN, there was no significant difference in the BK levels before and after surgery. (C) ROC curves were depicted and the AUCs of these biomarkers were calculated. The AUC of BK alone was 0.671 (95% CI: 0.590-0.751) and SCCA was 0.657 (95% CI: 0.579-0.735). Combined both, the AUC was 0.752 (95% CI: 0.682-0.821). ^*P<0.05, ^**P<0.01 and ^***P<0.001, n.s. non-statistical significance. BK, bradykinin; CC, cervical cancer; CIN, cervical intraepithelial neoplasia; SCCA, squamous cell carcinoma-related antigen.

Figure 2

BDKRB2 is overexpressed in cervical cancer tissues. (A) BDKRB2 was significantly upregulated in CC vs. normal samples in one dataset (Scotto Cervix Statistics) of the Oncomine online database (P<0.05). (B) The overexpression of BDKRB2 was further verified in CC tissues (n=15) and normal cervical tissues (n=9) by RT-qPCR (P<0.05). (C) Typical IHC images of BDKRB2 in CC, CIN and normal tissues. (D) The relevant cartogram of IHC scores of CC (n=44), CIN (n=12) and CON (n=8) tissues. BDKRB2 was notably overexpressed in CC (P<0.05) and CIN tissues (P<0.05) compared to normal cervical tissues. ^*P<0.05. n.s. non-statistical significance; BDKRB2, bradykinin B2 receptor; CC, cervical cancer; CIN, cervical intraepithelial neoplasia; NC, normal control.

Figure 3

BK promotes angiogenesis and upregulates VEGF expression in CC cells. (A) Representative images of tube formation experiment of HUVECs cultured with BK or HOE140. (B) The total tube length was quantified and statistics analysis was conducted. BK promoted angiogenesis and HOE140 played an inhibitory role. (C) The expression level of BDKRB2 was higher in the CC cell lines, SiHa and HeLa, and lower in the CIN cell line, S12. (D-F). BK upregulated the expression of VEGF in the SiHa and HeLa cells at the protein level and mRNA level, and in culture supernatants. HOE140 inversely suppressed VEGF expression. ^*P<0.05, ^**P<0.01 and ^***P<0.001, n.s. non-statistical significance. BK, bradykinin; CC, cervical cancer; CON, control; VEGF, vascular endothelial growth factor.

Figure 4

Overexpression or knockdown of BDKRB2 enhances or suppresses the expression of VEGF in CC cells, respectively. (A) BDKB2R overexpression and knockdown in the SiHa and HeLa cells was established and the transfection efficiency was examined by western blot analysis. A total of 10 μM of BK was added to the supernatant of all these cells. The corresponding VEGF expression was detected simultaneously. (B) The established cells were examined at the mRNA level. (C and D) BDKB2R overexpression in CC cells led to a higher VEGF expression and BDKB2R knockdown in CC cells led to a lower VEGF expression at the mRNA level and in culture supernatants. ^*P<0.05, ^**P<0.01 and ^***P<0.001. BDKRB2, bradykinin B2 receptor; CC, cervical cancer; VEGF, vascular endothelial growth factor.

Figure 5

BDKRB2 regulates tumor growth in tumor-bearing mice. (A) Survival rates of SiHa-oe (n=7), SiHa-con (n=6) and SiHa-sh (n=7) tumor-bearing mice over time. The survival time of SiHa-sh-B2R tumor-bearing mice was significantly longer than that of the SiHa-con group (P=0.024) and the SiHa-oe-B2R group (P=0.003). (B) Tumor growth volumes were measured and calculated in each group every week. Volume = ab²/2. (C) The corresponding fluorescence intensity was monitored every 2 weeks and quantitative analyzed with ROI tools of live Imaging software 4.0. (D) Typical fluorescence images were displayed at 4 weeks and 6 weeks following inoculation. (E) Representative IHC staining images of B2R, VEGF and CD31 in tumor sections from mice in the 3 groups. The black arrows indicate the neovascularization in the tumor margin. The mice bearing B2R-overexpressing tumors exhibited a significantly more rapid tumor growth, a shorter survival and more angiogenesis. ^*P<0.05 and ^**P<0.01.