Table 1.
Sequences of real-time RT-PCR primers.
Fig 1.
Effect of β-lapachone on the cell viability of metastatic melanoma cells.
(A and B) Cell viability of β-lapachone-treated B16F10 (A) and B16BL6 (B) cells. The cells were seeded at a density of 5 × 103 cells/well in 96-well microplates and then treated with various concentrations of β-lapachone for 24–72 h. After incubation, cell viability was determined by WST assay. Values are the means ± SD of data from three independent experiments. (C) Morphology of β-lapachone-treated cells. B16F10 and B16BL6 cells were treated with β-lapachone for 48 h. The morphological changes of the cells were observed under a phase-contrast microscope (magnification 200×).
Fig 2.
Effect of β-lapachone on cell cycle arrest in B16F10 cells.
(A) Cell cycle analysis on β-lapachone-treated B16F10 cells. The cells were treated with β-lapachone (1, 2, and 5 μM) for 48 h and fixed with 70% ethanol for at least 3 h. After centrifugation, cells were stained with PI solution and analyzed for determination of cell cycle phase distribution. Data are representative of three independent experiments. (B) The percentage of cells in each phase of the cell cycle. (C) mRNA expression of cyclin D1 and CDK4. B16F10 cells were treated with indicated concentrations of β-lapachone for 24 h. Values are the means ± SD of data from three independent experiments. *P < 0.05.
Fig 3.
Effect of β-lapachone on the apoptosis of B16F10 cells.
(A) β-Lapachone-induced apoptosis was observed by TUNEL assay under an inversion fluorescent microscope. B16F10 cells were incubated with indicated concentration of β-lapachone for 12 h. (B) B16F10 cells were incubated with the indicated concentrations of β-lapachone for 12 h and analyzed by Annexin V/7-AAD staining and flow cytometry. The figure is representative of three independent experiments. (C) The percentages of apoptotic cells were plotted. Values are the means ± SD of data from three independent experiments. *P < 0.05 and ***P < 0.001.
Fig 4.
Effect of β-lapachone on the apoptotic pathway in B16F10 cells.
A and B. B16F10 cells were treated with β-lapachone (5 μM) for 0–9 h (A) or various concentrations of β-lapachone for 12 h (B). Cell lysates were subjected to western blot analysis with PARP, caspase-3, -8, -9, Bcl-2, Bcl-xL, and Bax antibodies. (C and D) Relative levels of cleaved caspase-3 (c-cas-3), cleaved caspase-8 (c-cas-8), cleaved caspase-9 (c-cas-9), cleaved PARP (c-PARP), Bax, Bcl-2, and Bcl-xL were calculated using an Image J program. Values are the means ± SD of data from three independent experiments. *P < 0.05.
Fig 5.
Effect of β-lapachone on the MAPKs pathway in B16F10 cells.
(A) B16F10 cells were exposed to β-lapachone (5 μM) for 0–60 min and phosphorylation levels of MAPK proteins were determined by western blot analysis. (B and C) Role of MAPKs signaling on the β-lapachone-induced apoptosis in B16F10 cells. Cells were pretreated with the MAPK inhibitors SB203580 (5 μM, p38 inhibitor), U1026 (10 μM, ERK inhibitor), and SP600125 (10 μM, JNK inhibitor). After β-lapachone treatment for 24 h, cell viability was determined by WST assay (B) and Annexin V assay (C). (D) Expressions of caspase-3 and PARP in the β-lapachone and MAPK inhibitors SB203580, U1026, SP600125-treated B16F10 cells. (E) Relative levels of cleaved caspase-3 (c-cas-3) and cleaved PARP (c-PARP) were calculated using an Image J program. Values are the means ± SD of data from three independent experiments. Values are the means ± SD of data from three independent experiments. #P < 0.05 versus β-lapachone non-treated cells, *P < 0.05 versus only β-lapachone-treated cells.
Fig 6.
Effect of β-lapachone on the migration and invasion of B16F10 cells, through inhibition of MMP expression and activity.
(A) Cancer cell motility was determined by wound healing assay. Cells were cultured to 8–90% confluent monolayer in a 6-well plate and scratched with a 200 μl micropipet tip. After washing with a serum-free medium, the cells were treated for 24 h with the indicated concentrations of β-lapachone. The denuded zone was observed at 0 and 24 h and images were photographed using a microscope (100× magnification). (B) Invasion ability of B16F10 cells was determined by invasion assay using matrigel-coated transwell chamber. (C) The mRNA expression levels of MMP-2 and MMP-9 were measured by real-time RT-PCR after β-lapachone treatment for 24 h. (D) Inhibitory effect of β-lapachone on activity of MMP-2 and MMP-9. After 12 h starvation, cells were treated with β-lapachone for 24 h and conditioned medium was collected for use in gelatin zymography. Photographs are representative of three independent experiments. The band intensity was quantitatively analyzed using ImageJ software. Values are the means ± SD of data from three independent experiments. *P < 0.05 and **P < 0.01.
Fig 7.
Effect of β-lapachone on the EMT marker expression in B16F10 cells.
The mRNA expression levels of EMT-related genes were measured by real-time RT-PCR in β-lapachone-treated cells. B16F10 cells were treated with β-lapachone (0.01–1 μM) for 24 h. Values are the means ± SD of data from three independent experiments. *P < 0.05.
Fig 8.
Effect of β-lapachone on the lung metastasis of B16F10 cells.
C57BL/6 mice were intravenously injected with B16F10 cells (2 × 105 cells) via the tail vein. The mice were intraperitoneally injected with DMSO or β-lapachone (5 mg/kg) every other day until sacrifice. (A) Measurement of body weight during β-lapachone administration. (B) Lungs were excised and fixed with 3.7% formaldehyde to count and compare the patterns of pulmonary tumor nodule formation among the experimental groups. (C) The number of tumor nodules is expressed as the mean ± SD. Values are the means ± SD of data from three independent experiments. **P < 0.01.