β-Lapachone Inhibits Lung Metastasis of Colorectal Cancer by Inducing Apoptosis of CT26 Cells

Abstract

Background: β-Lapachone is a quinone-containing compound found in red lapacho ( Tabebuia impetiginosa, syn. T avellanedae) trees. Lapacho has been used in traditional medicine by several South and Central American indigenous people to treat various types of cancer. The purpose of this study was to investigate the antimetastatic properties of β-lapachone and the underlying mechanisms using colon cancer cells.

Methods: This research used metastatic murine colon cancer cell lines, colon 26 (CT26) and colon 38 (MC38). A WST assay, annexin V assay, cell cycle analysis, wound healing assay, invasion assay, western blot analysis, and real-time reverse transcription-polymerase chain reaction were performed to examine the effects of β-lapachone on metastatic phenotypes and molecular mechanisms. The effect of β-lapachone on lung metastasis was assessed in a mouse experimental metastasis model.

Results: We found that the inhibition of proliferation of the colon cancer cell lines by β-lapachone was due to the induction of apoptosis and cell cycle arrest. β-Lapachone induced the apoptosis of CT26 cells through caspase-3, -8, and -9 activation; poly(ADP-ribose) polymerase cleavage; and downregulation of the Bcl-2 family in a dose- and time-dependent manner. In addition, a low concentration of β-lapachone decreased the cell migration and invasion by decreasing the expression of matrix metalloproteinases-2 and -9, and increased the expression of tissue inhibitors of metalloproteinases-1 and -2. Moreover, β-lapachone treatment regulated the expression of epithelial-mesenchymal transition markers such as E- and N-cadherin, vimentin, β-catenin, and Snail in CT26 cells. In the mouse experimental metastasis model, β-lapachone significantly inhibited the lung metastasis of CT26 cells.

Conclusions: Our results demonstrated the inhibitory effect of β-lapachone on colorectal lung metastasis. This compound may be useful for developing therapeutic agents to treat metastatic cancer.

Keywords: CT26 cells; apoptosis; colorectal cancer; lung metastasis; β-lapachone.

Figure 1.

β-Lapachone decreases viability of metastatic murine colon carcinoma cells. (A) Chemical structure of β-lapachone. (B and C) Viability of β-lapachone-treated CT26 and MC38 cells. Cells were seeded at a density of 3 × 10³ cells/well in 96-well microplates and then treated with various concentrations of β-lapachone for 24 and 48 hours. After incubation at 37°C, cell viability was determined using the WST assay. Results are expressed as the mean ± SD of 3 independent experiments. **P < .01, ***P < .001. (D) Morphology of β-lapachone-treated CT26 cells. After 24 hours of incubation with β-lapachone, photographs were acquired by microscopy. The photographs are representative of 3 independent experiments.

Figure 2.

β-Lapachone induces apoptosis through extrinsic and intrinsic signaling pathways in CT26 cells. (A) CT26 cells were incubated with the indicated concentrations of β-lapachone for 9 hours and stained with annexin V and PI. The figure is representative of 3 independent experiments. (B) CT26 cells were treated with β-lapachone (1 µM) for 0 to 9 hours. (C) CT26 cells were treated with various concentrations of β-lapachone for 9 hours and subjected to western blotting with antibodies against PARP, caspase-3, -8, -9, Bcl-2, Bcl-xL, and Bax.

Figure 3.

β-Lapachone induces G0/G1 phase cell cycle arrest through inhibition of cyclin D1 and CDK4 expression. (A) Cell cycle analysis of CT26 cells after treatment with β-lapachone for 24 hours. Data are representative of 3 independent experiments. (B) Percentages of cells with the DNA content consistent with each phase of the cell cycle were plotted. (C) mRNA expression of cyclin D1 and CDK4. CT26 cells were treated with various concentrations of β-lapachone for 24 hours. Results are expressed as the mean ± SD of 3 independent experiments. *P < .05.

Figure 4.

β-Lapachone regulates mRNA expression levels of EMT markers. mRNA expression levels of EMT markers were analyzed by real-time RT-PCR after treatment of CT26 cells with β-lapachone (0-100 nM) for 24 hours. (A) Epithelial marker; E-cadherin. (B-E) Mesenchymal markers; N-cadherin, vimentin, β-catenin, and Snail. Results are expressed as the mean ± SD of 3 independent experiments. *P < .05 and **P < .01.

Figure 5.

β-Lapachone reduces migration and invasion ability. (A) Wound healing assay. CT26 cells were cultured to 80% confluence in a 6-well plate, and the cell layer was scratched with a 200 µL micropipette tip. After washing with a serum-free medium, the cells were treated for 24 hours with the indicated concentrations of β-lapachone. After the incubation, images were photographed using a microscope (100× magnification). (B) Invasion assay. CT26 cells (5 × 10⁴ cells/well) were cultured in a 24-well plate with a matrigel-coated transwell chamber. After 24 hours of incubation with β-lapachone, the inner side of the chamber was fixed and stained. Photographs are representative of 3 independent experiments. (C) MMP-2 and MMP-9 activities in β-lapachone-treated CT26 cells were determined by gelatin zymography. (D and E) mRNA expression levels of the cell invasion–related factors MMP-2, -9 (D) and TIMP-2, -1 (E) were analyzed by real-time RT-PCR after β-lapachone (10 or 100 nM) treatment for 24 hours. Results are expressed as the mean ± SD of 3 independent experiments. *P < .05 and **P < .01.

Figure 6.

β-Lapachone inhibits colorectal lung metastasis. BALB/c mice were intravenously transplanted with 1 × 10⁵ cells of CT26 cancer cells into the tail vein. Then, the mice were divided into 3 groups (n = 6) and subjected to intraperitoneal injection of β-lapachone (2 or 5 mg/kg) once every 2 days until sacrifice. The control group mice were administered the same volume of DMSO. (A) The body weight of the mice was monitored for assessing toxicity. (B) Lungs were excised and stained with Bouin’s solution to compare the patterns of pulmonary tumor nodule formation among the experimental groups. (C and D) The number of tumor nodules (C) and lung weight (D) are expressed as the mean ± SD. (E and F) mRNA expression levels of EMT markers (E) and MMP-2/9 (F) were measured in lung tissues. *P < .05, **P < .01, and ***P < .001. Data are representative of 3 independent experiments.