Fig 1.
Synthetic approaches for the production of PNAP-1–PNAP-8 and PNAP-2h–PNAP-8h.
The eight 2-phenylnaphthalenes (PNAP-1–PNAP-8) were easily obtained from phenylacetonitriles and benzaldehydes through six steps. Subsequently, the six hydroxy-PNAPs (PNAP-2h−PNAP-7h) were obtained by demethylation of the corresponding methoxy-PNAPs (PNAP-2−PNAP-7). Unfortunately, attempts at the demethylation of PNAP-8 under the same conditions led to complex mixtures. Another hydrophilic amino-PNAP (PNAP-8h) was produced by the reduction of nitro-PNAP (PNAP-8).
Table 1.
Effect of 2-Phenylnaphthalenes PNAP-1−PNAP-8, Their Demethylation Derivatives PNAP-2h−PNAP-7h, and Amino PNAP-8h on the Viability of MCF-7 Cells.
Fig 2.
Effect of PNAP-1, -3h, and -6h on MCF-7 morphology.
MCF-7 cells were treated with PNAP-1, -3h, and -6h at three concentrations (5, 10, and 25 μM) for 48 h, and then, (A–J) the morphology was observed by light microscopy (100×). The cells were stained with Hoechst 33342, and then, (K–T) the morphology was observed by fluorescence microscopy (200×). (A/K) control; (B/L) 5 μM PNAP-1; (C/M) 10 μM PNAP-1; (D/N) 25 μM PNAP-1; (E/O) 5 μM PNAP-3h; (F/P) 10 μM PNAP-3h; (G/Q) 25 μM PNAP-3h; (H/R) 5 μM PNAP-6h; (I/S) 10 μM PNAP-6h; and (J/T) 25 μM PNAP-6h.
Fig 3.
Effect of PNAP-1, -3h, and -6h on MCF-7 cell cycle progression.
MCF-7 cells were treated with PNAP-1, -3h, and -6h at three concentrations (5, 10, and 25 μM) for 48 h, and then, (A) the cells were fixed and stained with propidium iodide to analyze the DNA content by flow cytometry. (B, C) Cell cycle-associated proteins (cyclin D1, CDK4, cyclin E, CDK2, p21, p27, cyclin B1, and CDK1) were detected and analyzed by western blot. The expression was quantified with the computerized Gel-Pro Analyzer image analysis system. The data are expressed as the means ± SEM from three independent assays. *P<0.05 and **P<0.01 are significantly different from the control.
Fig 4.
Effect of PNAP-1, -3h, and -6h on the expression of apoptosis-related proteins.
(A) MCF-7 cells were treated with PNAP-1, -3h, and -6h at three concentrations (5, 10, and 25 μM) for 48 h, and the expression of cleaved caspase-7, -8, -9 and PARP was determined by western blotting. (C) MCF-7 cells were treated with 25 μM PNAP-1, -3h, and -6h at three concentrations (5, 10, and 25 μM) for 48 h, and the Bcl-2, Bcl-xl, Bax, Bax/Bcl-2, Bid, and Fas levels were then detected by western blot. (B, D) The expression was quantified using a computerized Gel-Pro Analyzer image analysis system. The data are expressed as the means ± SEM from three independent assays. *P<0.05 and **P<0.01 are significantly different from the control.
Fig 5.
Effect of PNAP-6h on the expression of caspase-7, -8, and -9 proteins.
MCF-7 cells were incubated in the presence or absence of (A) the caspase-8 inhibitor z-IETD-FMK or (B) the caspase-9 inhibitor z-LEHD-FMK for 1.5 h before the addition of PNAP-6h. Western blot analyses were conducted with antibodies to caspase-7, -8, and -9, and actin. (C) MCF-7 cells were treated with PNAP-6h in the presence or absence of caspase-8 inhibitor, or caspase-9 inhibitor, and the morphology was then observed by light microscopy.
Fig 6.
Expression of MAPKs in PNAP-induced apoptosis.
(A) MCF-7 cells were treated with 25 μM PNAP-1, -3h, and -6h at three time points (12, 24, and 48 h). (B) MCF-7 cells were treated with PNAP-1, -3h, and -6h at three concentrations (5, 10, and 25 μM) for 48 h. ERK, p38, and JNK, as well as their respective phosphorylated forms, were then analyzed by western blot. The data are expressed as the means ± SEM from three independent assays. *P<0.05 and **P<0.01 are significantly different from the control.
Fig 7.
Proposed model of PNAP-6h-mediated MCF-7 cell cycle arrest and apoptosis.
PNAP-6h causes cell cycle arrest at the S and G2/M phases. PNAP-6h modulates MCF-7 apoptosis via activation of the intrinsic/extrinsic pathways and the p38/ERK pathway.