Berberine enhances radiosensitivity of esophageal squamous cancer by targeting HIF-1α in vitro and in vivo

Figures & data

Figure 1. Berberine sensitizes hypoxic ESCC cancer cells to IR. (A) Berberine inhibited growth of ESCC cells in a time- and dose-dependent manner. (B) Clonogenic assay showing that ESCC cancer cells became radioresistant in hypoxia conditions and that was reversed by berberine. (C and D) Flow cytometric analysis showing that berberine induced apoptosis of hypoxic ECA109 cells. (a) normoxia IR; (b) hypoxia IR; (c) hypoxia IR Ber 5 μM; (d) hypoxia IR Ber 15 μM. The bars presented means ± s.d. of three separate experiments. norm, normoxia; hypo, hypoxia; ber, berberine; DDP, cisplatin. ***P < 0.001; **P < 0.01; *P < 0.05.

Table 1. The radiosensitization activity of berberine in hypoxia ECA109 cells

	D0	Dq	SF2	SER
Hypoxia	4.56	2.01	0.82
Normoxia	2.58	0.98	0.47	1.76
Hypo+Ber 5 μM	3.24	1.91	0.80	1.40
Hypo+Ber 15 μM	2.94	1.43	0.63	1.55
DDP	2.33	0.81	0.41	1.95

Hypo, hypoxia; Ber, berberine; DDP, cisplatin.

Table 2. The radiosensitization activity of berberine in hypoxia TE13 cells

	D0	Dq	SF2	SER
Hypoxia	4.62	2.09	0.84
Normoxia	3.27	0.91	0.45	1.41
Hypo+Ber 5 Mm	3.84	1.84	0.77	1.20
Hypo+Ber 15 Mm	2.99	1.43	0.63	1.54
DDP	2.50	0.72	0.39	1.85

Hypo, hypoxia; Ber, berberine; DDP, cisplatin.

Figure 2. Berberine inhibits HIF-1α and VEGF protein expression in ESCC cells. (A and B) Hypoxia stimulated HIF-1α and VEGF expression in ESCC cell lines. a: ECA109; b: TE13. (C and D) The expression of HIF-1α and VEGF was inhibited by berberine in ESCC cells. c: ECA109; d: TE13. β-actin was loading control.

Figure 3. Berberine inhibits HIF-1α and VEGF protein expression in ESCC in vivo. (A) The nuclear location of HIF-1α was observed by laser scanning confocal microscopy using HIF-1α antibody. Representative images showing nuclear colocalization of HIF-1α (green stain) in cells under hypoxia in the presence or absence of berberine (5 μM and 15 μM). (B) Imumunohistochemical staining of HIF-1α and VEGF in tumor tissues of ECA109 xenograft bearing nude mice.

Figure 4. Berberine sensitizes ESCC to IR in vivo. ECA109 xenograft bearing male BALB/c mice were divided into four treatment groups (n = 6): control; berberine alone; irradiation alone; the combination of berberine and irradiation. 10 days after inoculation of ECA109 cells (1 × 10⁶ cells/mouse), the mice were treated with 8 Gy single fraction irradiation. (A) Measurement of tumor size. Data represented average tumor volume; error bars, SD. (B) Representative images of ECA109 xenograft bearing mice.

Table 3. Effect of berberine on response of ECA109 xenografted tumor to irradiation

Treatment	Doubling time(days)	Absolute growth delay (days)^a	Normalized growth delay (days)^b	Enhancement factor
Control	3.8 ± 0.1
Berberine	4.0 ± 0.1	0.2 (4.0–3.8)
IR	6.7 ± 0.3	2.9 (6.7–3.8)
IR + berberine	14.1 ± 0.8	10.3 (14.1–3.8)	10.1 (10.3–0.2)	3.5 (10.1/2.9)

^a Absolute growth delay, the doubling tumor time of treatment group minus that of control group; ^bNormalized growth delay, the time of absolute growth delay of tumor in IR combined with berberine group minus that of berberine group.