Apoptotic induction and inhibition of NF-κB signaling pathway in human prostatic cancer PC3 cells by natural compound 2,2′-oxybis (4-allyl-1-methoxybenzene), biseugenol B, from Litsea costalis: an in vitro study

Figures & data

Figure 1 Structures of compound 2,2′-oxybis (4-allyl-1-methoxybenzene) or biseugenol B.

Figure 2 MTT assay growth curve of PC3 cells treated with biseugenol B at 24, 48, and 72 hours.

Abbreviation: MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Table 1 IC₅₀ concentration of biseugenol B

Cell line	IC50 (µg/mL)
	24 hours	48 hours	72 hours
PC3	2.42±1.05	2.1±0.38	1.33±0.47
RWPE-1	57.43±1.15	45.65±2.94	34.92±1.6

Note: Data presented as mean ± standard deviation.

Abbreviation: IC₅₀, half maximal inhibitory concentration.

Figure 3 AO-PI double-staining cell morphological analysis in untreated and treated PC3 cells with biseugenol B.

Notes: Normal structure without noticeable apoptosis or necrosis is shown in untreated cells (A). After treatment with 2 µg/mL, EA features were observed with intercalated AO (bright green) among the fragmented DNA (B). In 4 µg/mL treatment, the hallmark of LA was detected, which is represented by blebbing and orange color (C). After treatment with 8 µg/mL, SN was visible by bright red color (D).
Abbreviations: AO, acridine orange; VI, viable cells; EA, early apoptosis; LA, late apoptosis; SN, secondary necrosis; PI, propidium iodide.

Figure 4 The percentages of VI, EA, LA and SN cells after biseugenol B treatment.

Notes: A significant increase was observed in PC3 cell death via apoptosis in a dose-dependent manner. The results are shown as mean ± SD of three independent experiments. *P<0.05. **P<0.005.
Abbreviations: VI, viable cells; EA, early apoptosis; LA, late apoptosis; SN, secondary necrosis; SD, standard deviation.

Figure 5 The effect of biseugenol B on EA and LA of PC3 cells.

Notes: PC3 cells were treated with different concentrations of biseugenol B and maintained for 24 hours at 37°C in a CO₂ incubator. The cells were analyzed after staining with FITC-conjugated AV and PI by flow cytometer. Untreated cells served as control (A). AV⁺/PI⁻ represents the EA events shown in lower right quadrant (Q4-1). The late stage of apoptosis/dead cells (AV⁺/PI⁺) is shown in quadrant Q2-1. The effects of 2, 4 and 8 µg/mL exposures of PC3 cells to biseugenol B (B–D). Bar chart representing the percentage of VI, EA, LA and necrotic cells in different concentrations of treatment with biseugenol B on PC3 cells (E). The results are shown as mean ± SD of three independent experiments. *P<0.05. **P<0.005.
Abbreviations: EA, early apoptosis; LA, late apoptosis; FITC, fluorescein isothiocyanate; AV, annexin V; PI, propidium iodide; VI, viable cells; SD, standard deviation.

Figure 6 Cell cycle histogram from analyses of PC3 cells treated with 0 µg/mL (A), 2 µg/mL (B), 4 µg/mL (C) and 8 µg/mL (D) of biseugenol B for 24 hours. (E) Summary of cell cycle progression for control and biseugenol B-treated PC3 cells.

Notes: The results are shown as mean ± SD of three independent experiments. *P<0.05. **P<0.005.
Abbreviation: SD, standard deviation.

Table 2 Effect of biseugenol B on cell cycle phases

Concentration	Sub G0–G1	G0–G1	S	G2/M
0 µg/mL	1.94±0.32	58.84±1.15	16.45±2.02	8.81±1.65
2 µg/mL	2.47±0.46	66.88±1.58**	18.73±1.38*	10.22±1.06**
4 µg/mL	1.75±0.42	78.06±1.37**	7.77±0.94**	11.22±1.67**
8 µg/mL	24.73±0.24**	64.93±1.04**	3.59±0.55**	5.94±1.48**

Notes: Human prostate cancer cells were exposed to biseugenol B at various concentrations (0, 2, 4, and 8 µg/mL) for 24 hours. The table summarizes the percentages of cells in each phase of the cell cycle after treatment with biseugenol B. Data in the same vertical column but different rows refer to the same phase of the cell cycle and different biseugenol B concentrations. The results are shown as mean ± SD of three independent experiments.

* P<0.05.

** P<0.005.

Abbreviation: SD, standard deviation.

Figure 7 The effect of biseugenol B on nuclear size, MMP, cell membrane permeability, and cytochrome c release in PC3.

Notes: (A) Representative images of the untreated PC3 cells and PC3 cells treated with 4 µg/mL biseugenol and stained with Hoechst for nucleus, cytochrome c, membrane permeability and MMP dyes and cytochrome c dye. The images from each row are obtained from the same field of the same treatment sample (magnification ×20). (B) The bar chart represents the average fluorescence intensities of Hoechst, cell permeability dye, MMP, and cytochrome c in untreated and treated PC3 cells with biseugenol B. Data are mean ± SD of fluorescence intensity readings measured from different photos taken. *P<0.05. **P<0.005.
Abbreviations: MMP, mitochondrial membrane potential; SD, standard deviation.

Figure 8 Relative bioluminescence expression of caspase-3/7, caspase-8, and caspase-9 in PC3 and RWPE-1 cells treated with biseugenol B.

Notes: (A) Relative bioluminescence expression of caspase-3/7, caspase-8 and caspase-9 in PC3 cells treated with biseugenol B in different concentrations. The results are shown as mean ± SD of three independent experiments. *P<0.05. **P<0.005. (B) Relative bioluminescence expression of caspase-3/7, caspase-8 and caspase-9 in RWPE-1 cells treated with biseugenol B in different concentrations. The results are shown as mean ± SD of three independent experiments. (C) Comparison of relative bioluminescence expression of caspase-3/7, caspase-8, and caspase-9 between PC3 and RWPE-1 cells treated with biseugenol B in different concentrations.
Abbreviation: SD, standard deviation.

Figure 9 DCF-fluorescence intensity after exposure of biseugenol B for 24 hours in PC3 and RWPE1.

Notes: (A) Effects of biseugenol B on PC3 cells in ROS production. DCF-fluorescence intensity after 0, 1, 2, 4 and 8 µg/mL of biseugenol B exposure at 24 hours. The results are shown as mean ± SD of three independent experiments. **P<0.005. (B) Effects of biseugenol B on RWPE-1 cells in ROS production. DCF-fluorescence intensity after 0, 1, 2, 4 and 8 µg/mL of biseugenol B exposure at 24 hours. The results are shown as mean ± SD of three independent experiments. (C) Effects of biseugenol B on PC3 and RWPE-1 cells in ROS production. The results are shown as mean ± SD of three independent experiments.
Abbreviations: ROS, reactive oxygen species; DCF, dichlorodihydrofluorescein; SD, standard deviation.

Figure 10 Effects of biseugenol B on the Bax, Bcl-2 and Hsp70 mRNA expression level in PC3 cells.

Notes: PCR analysis of biseugenol B in selected apoptotic signaling markers. The blot densities are expressed as fold of control (A). The bar chart represents dose-dependent downregulation of Bax (B). The bar charts represent dose-dependent increase of Bcl-2 (C) and Hsp70 (D). The results are shown as mean ± SD of three independent experiments. *P<0.05. **P<0.005.
Abbreviations: Bax, Bcl-2-associated X; Bcl-2, Bcl-cell lymphoma-2; Hsp70, heat-shock protein 70; PCR, polymerase chain reaction; SD, standard deviation.

Figure 11 Western blot analysis of biseugenol B in the selected apoptotic signaling markers.

Notes: The blot densities are expressed as fold of control (A). Bar chart represents dose-dependent downregulation of Bax (B). Bar chart represents dose-dependent increase of Bcl-2 (C) and Hsp70 (D). The results are shown as mean ± SD of three independent experiments. *P<0.05. **P<0.005.
Abbreviations: Bax, Bcl-2-associated X; Bcl-2, Bcl-cell lymphoma-2; Hsp70, heat-shock protein 70; SD, standard deviation.

Figure 12 Inhibition of TNF-α-induced NF-κB nuclear translocation by biseugenol B.

Notes: Photographs of intracellular targets in stained PC3 cells treated with biseugenol B for 3 hours (A) and then stimulated for 30 minutes with TNF-α 10 ng/mL (NF-κB activation) (B). Decline in average fluorescent intensity of nuclei NF-κB, confirming that biseugenol B inhibited TNF-α-induced translocation of NF-κB from the cytoplasm to the nucleus (C).
Abbreviations: TNF-α, tumor necrosis factor-alpha; NF-κB, nuclear factor kappa-B.

Figure 13 Immunoblot analysis of nuclear NF-κB.

Notes: (A) Immunoblot analysis of nuclear NF-κB p65. I, untreated PC3; II, stimulated with TNF-α only; III, IV and V, PC3 treated with 2, 4 and 8 µg/mL of biseugenol B, respectively; VI, PC3 treated with curcumin. (B) Representative bar chart indicating a significant decline of nuclear NF-κB p65 expressed as folds of control. The results are shown as mean ± SD of three independent experiments. **P<0.005.
Abbreviations: NF-κB, nuclear factor kappa-B; SD, standard deviation.