Lithium chloride suppresses colorectal cancer cell survival and proliferation through ROS/GSK-3β/NF-κB signaling pathway

Abstract

Glycogen synthase kinase-3β (GSK-3β), a serine/threonine protein kinase, has been regarded as a potential therapeutic target for multiple human cancers. In addition, oxidative stress is closely related to all aspects of cancer. We sought to determine the biological function of lithium, one kind of GSK-3β inhibitors, in the process of reactive oxygen species (ROS) production in colorectal cancer. In this study, we analyzed the cell apoptosis and proliferation by cell viability, EdU, and flow cytometry assays through administration of LiCl. We used polymerase chain reaction and Western blotting to establish the effect of GSK-3β inhibition on the nuclear factor-κB (NF-κB) pathway. Results showed administration of LiCl increased apoptosis and the level of ROS in colorectal cancer cells. Furthermore, the underlying mechanisms could be mediated by the reduction of NF-κB expression and NF-κB-mediated transcription. Taken together, our results demonstrated that therapeutic targeting of ROS/GSK-3β/NF-κB pathways may be an effective way for colorectal cancer intervention, although further preclinical and clinical testing are desirable.

Figures

Figure 1

(a) Morphological changes of SW480 cells exposed to different concentrations of LiCl (10 mM, 20 mM, 40 mM, and 60 mM). (b) The percentage of viable SW480 cells. Cells were treated with different concentrations of LiCl (10 mM, 20 mM, 40 mM, and 60 mM) or treated with LiCl for 6, 12, 24, or 48 hours, respectively. Each point is mean ± SEM for at least three individual experiments. Original magnification, ×100.*P < 0.05 and **P < 0.01 versus Control group.

Figure 2

LiCl inhibited proliferation in SW480 cells. Cell proliferation assay was preformed, in which EdU-labeled proliferative cells (red) and Hoechst-stained nuclei (blue) were observed under a fluorescent microscope. Cells were treated with vehicles (PBS) and different concentrations of LiCl (10 mM, 20 mM, 40 mM, and 60 mM), respectively. Data are representative of at least three independent experiments and are expressed as the mean ± SEM. Original magnification, ×100. *P < 0.05 and **P < 0.01 versus Control group.

Figure 3

Quantification of early and late apoptosis in SW480 cells treated with different concentrations of LiCl for 24 h was determined by flow cytometry. Representative results were from three independent experiments. Data were shown as mean ± SEM. *P < 0.05 and **P < 0.01 versus Control group.

Figure 4

SW480 cells were loaded with a fluorescent probe H2DCF-DA (10 μM) for 20 minutes and observed under fluorescence microscopy. Representative photomicrographs showing ROS within the cytoplasm of cells and merged images showing cell morphology. Fluorescent signals were quantified using a fluorometer at excitation and emission wavelengths of 488 nm and 520 nm, respectively. *P < 0.05 and **P < 0.01 versus Control group. Original magnification, ×200.

Figure 5

Expression of GSK-3β and presence of phosphor-GSK-3β(Ser9) (inactive form) and phosphor-GSK-3β(Tyr216) (active form) were detected in extracts of SW480 cells treated with LiCl. The amount of protein extract in each sample was monitored by expression of β-actin.

Figure 6

Effect of LiCl on the expression of NF-κB, Bal-2, and survivin protein and mRNA levels. (a) The protein expression of NF-κB, Bal-2, and survivin. (b-c) The mRNA levels of NF-κB, Bal-2, and survivin were analyzed by real-time PCR. The SW480 cells treated with vehicles (PBS) and different concentrations of LiCl (10 mM, 20 mM, 40 mM, and 60 mM), respectively. Data are expressed as mean ± SEM for at least three individual experiments. *P < 0.05 and **P < 0.01 versus Control group.