Anti-cancer activity and cellular uptake of 7,3′,4′- and 7,8,4′-trihydroxyisoflavone in HepG2 cells under hypoxic conditions

Figures & data

Figure 1. Chemical structure of (A) Daidzein, (B) 734THIF and (C) 784THIF.

Table 1. Primary antibodies used.

Primary antibody	Dilution ratio	Item number and manufacturer
HIF-1α	1:1000	#3716, Cell Signalling Technology, Danvers, MA, USA
CAV-1	1:1000	610057, Biosciences, Franklin Lakes, NJ, USA
VEGF	1:1000	19003-1-AP, Proteintech Group, Inc., Wuhan, China
COX-2	1:1000	#12282, Cell Signalling
NRF-2	1:1000	#12721, Cell Signalling
SIRT1	1:1000	#9475, Cell Signalling
E-cadherin	1:1000	ARG66195, Arigo Biolaboratories Corp., Hsinchu, Taiwan
N-cadherin	1:1000	ARG23870, Arigo
Vimentin	1:1000	ARG66199, Arigo
phospho-ERK	1:1000	#05-797R, Merck Millipore
phospho-p38	1:1000	#09-272, Merck Millipore
BCL-2	1:1000	#4223, Cell Signalling
MMP-9	1:1000	ARG54980, Arigo
GAPDH	1:2000	sc47724, Santa Cruz Biotechnology, Dallas, TX, USA

Figure 2. Three-dimensional (3D) and two-dimensional (2D) schematic diagrams of the docking poses of celecoxib (A), daidzein (B), 734THIF (C), and 784THIF (D) within the COX-2 active site (PDB code 3LN1). Green structures in the 3D plot: the analysed compounds.

Table 2. Docking score and binding energy of daidzein, 734THIF, 784THIF, celecoxib, and sorafenib with COX-2 and VEGFR2.

Compounds	Proteins	Docking score^a (kcal mol^−1)	Binding energy (kcal mol^−1)
daidzein	COX-2	−31.44	−30.91
734THIF		−31.20	−41.16
784THIF		−31.43	−41.69
Celecoxib		−20.62	−32.44
daidzein	VEGFR2	−25.65	−44.02
734THIF		−28.96	−63.37
784THIF		−26.61	−60.23
Sorafenib		−45.01	−94.20

^a The docking score was evaluated using the CDOCKER method in DS software.

Figure 3. Three-dimensional (3D) and two-dimensional (2D) schematic diagrams of the docking poses of sorafenib (A), Daidzein (B), 734THIF (C), and 784THIF (D) within the VEGFR2 active site (PDB code 4ASD). Green structures in the 3D plot: the analysed compounds.

Figure 4. Viability of HepG2 cells treated with 734THIF (A) and 784THIF (B) under different oxygen conditions. HepG2 cell viability was analysed using MTT. n = 3 in each group; *P < 0.05 compared with 0 h normoxic control; ^#P < 0.05 compared with other conditions.

Figure 5. Expression of hypoxia-related proteins in HepG2 cells. (A), (C), and (E) indicate the expression of HIF1a, VEGF, and CAV-1, respectively, under post-hypoxic conditions; (B), (D), and (F) indicate the expression of HIF1a, VEGF, and CAV-1, respectively, under pre-hypoxic conditions. n = 3 in each group; ^#P < 0.05 compared with normoxic control; * P < 0.05 compared with hypoxic control; ^$P < 0.05 734THIF compared with 784THIF.

Figure 6. Expression of a pro-inflammatory protein and oxidative homeostasis-related proteins in HepG2 cells. (A), (C), and (E) indicate the expression of COX-2, NRF-2, and SIRT-1, respectively, under post-hypoxic conditions; (B), (D), and (F) indicate the expression of COX-2, NRF-2, and SIRT-1, respectively, under pre-hypoxic conditions. n = 3 in each group; ^#P < 0.05 compared with normoxic control; * P < 0.05 compared with hypoxic control; ^$P < 0.05 734THIF compared with 784THIF.

Figure 7. Expression of EMT makers in HepG2 cell. (A), (C), and (E) indicate the expression of E-cad, N-cad, and vimentin, respectively, under post-hypoxic conditions; (B), (D), and (F) indicate the expression of E-cad, N-cad, and vimentin, respectively, under pre-hypoxic conditions. n = 3 in each group; ^#P < 0.05 compared with normoxic control; * P < 0.05 compared with hypoxic control; ^$P < 0.05 734THIF compared with 784THIF.

Figure 8. Expression of MMP-9 in and migration rate of HepG2 cells treated with 734THIF and 784THIF. The expression of MMP-9 under post-hypoxic (A) and pre-hypoxic conditions (B), respectively. Wound healing assay (C) and its closure percentage at different time points (D). n = 3 in each group; ^#P < 0.05: compared with normoxic control; *P < 0.05: compared with hypoxic control; ^$P < 0.05: 734THIF compared with 784THIF.

Figure 9. Expression of MAPKs (p-ERK and p-p38) and anti-apoptosis proteins in HepG2 cells. (A), (C), and (E) indicate the expression of p-ERK, p-p38, and BCL-2, respectively, under post-hypoxic conditions; (B), (D), and (F) indicate the expression of p-ERK, p-p38, and BCL-2, respectively, under pre-hypoxic conditions. n = 3 in each group; #P < 0.05 compared with normoxic control; * P < 0.05 compared with hypoxic control; $P < 0.05 734THIF compared with 784THIF.

Table 3. Percentage of protein expression inhibition by 734THIF and 784THIF under different hypoxic conditions.

Protein	Inhibition rate (%)^a
	Post-hypoxia		Pre-hypoxia
	734THIF	784THIF	734THIF	784THIF
HIF-1α	NI	50.87 ± 11.55	NI	NI
VEGF	25.55 ± 3.37	40.25 ± 10.79	NI	18.62 ± 4.55
CAV-1	11.52 ± 27.11	23.48 ± 31.47	22.81 ± 15.84	74.01 ± 11.24^b
COX-2	53.15 ± 7.88	57.89 ± 9.77	28.72 ± 9.18	67.73 ± 6.52^b
NRF-2	39.01 ± 5.04	44.52 ± 5.56	NI	NI
SIRT1	NI	NI	18.61 ± 14.33	16.91 ± 9.83
E-cad	NI	NI	NI	NI
N-cad	26.02 ± 8.13	45.16 ± 4.49^b	38.14 ± 8.71	53.86 ± 6.71
Vimentin	NI	22.81 ± 7.66	NI	20.57 ± 1.48
MMP-9	29.34 ± 1.81	65.21 ± 4.03^b	39.15 ± 13.27	41.30 ± 15.48
p-ERK	NI	NI	NI	NI
p-p38	NI	NI	NI	NI
BCL-2	64.98 ± 10.28	72.21 ± 7.17	52.42 ± 20.14	77.33± 1.89

NI represents “No Inhibition” of hypoxia-induced protein expression.

^a Inhibition rate = 1 - (drug group fold value/hypoxia-induced group fold value) × 100%

^b Indicates a significant difference in the expression rate of 734THIF inhibitory protein under the same hypoxic conditions.

Table 4. 734THIF and 784THIF levels in HepG2 cells under normoxic and different hypoxic conditions.

Condition	Normoxia (h)	Hypoxia (h)	Intracellular level (ng)
			734THIF	784THIF
Normoxia	6	0	81.12 ± 3.34	52.87 ± 1.86*
	12	0	17.90 ± 0.95	33.75 ± 0.81*
Hypoxia	0	6	135.26 ± 9.97	50.03 ± 1.71*
Post-hypoxia	6	6	26.89 ± 4.06	37.29 ± 0.69*
Pre-hypoxia	6	6	21.56 ± 2.34	33.72 ± 3.09*

Values are presented as mean ± SD (n = 3).

*P < 0.05: compared with 734THIF level.

Data availability statement

The data that support the findings of this study are available from the corresponding author on reasonable request. Some data may not be made available because of privacy or ethical restrictions.