Functional disruption of macrophage migration inhibitory factor (MIF) suppresses proliferation of human H460 lung cancer cells by caspase-dependent apoptosis

Abstract

Background: Macrophage migration inhibitory factor (MIF) is important in regulating cell proliferation and apoptosis in both normal and cancerous cells, and may be important in cancer progression and metastasis. In human non-small cell lung cancer (NSCLC), the underlying mechanisms responsible for MIF-dependent regulation of cellular proliferation, and cell death remain poorly appreciated.

Methods: The human H460 lung cancer cell-line was treated with an optimally determined dose of 50 pmol/ml MIF siRNA, following which cell proliferation, cell cycle and apoptosis were analyzed. Additionally, known pathways of apoptosis including expression of Annexin-V, enhanced production of caspases-3 and -4 and expression of the Akt signaling protein were assessed in an attempt to provide insights into the signaling pathways involved in apoptosis following disruption of MIF expression.

Results: Specific siRNA sequences markedly decreased MIF expression in H460 cells by 2 to 5-fold as compared with the negative control. Moreover, MIF miRNA dampened not only cellular proliferation, but increased the frequency of apoptotic cells as assessed by cell-surface Annexin-V expression. Entry of cells into apoptosis was partly dependent on enhanced production of caspases -3 and -4 while not affecting the expression of either caspase-8 or the Akt signaling pathway.

Conclusions: In a model of NSCLC, knockdown of MIF mRNA expression dampened H460 proliferation by mechanisms partly dependent on entry of cells into apoptosis and enhanced production of caspase-3 and -4. MIF expression may thus be important in NSCLC progression. Targeting MIF may have clinical utility in the management of human lung cancer.

Figure 1

Expression of MIF protein by Western immunoblot analysis in H460 and A549 cells. A549 cells showed a weaker signal for MIF expression as compared with H460 cells normalized to expression of β-actin. For this reason, we selected the H460 cell-line as a model of human non-small cell lung cancer in all subsequent experiments.

Figure 2

Dose-dependent detection of siRNA transfection efficacy by immunofluorescence observation. The following doses of siRNA were assessed: A: 30 pmol/ml. B: 50 pmol/ml. C: 70 pmol/ml. D: 100 pmol/ml. By visual inspection of the fluorescence intensity and frequency of cells transfected, we selected a dose of 50 pmol/ml in subsequent experiments.

Figure 3

siRNA-mediated knockdown of MIF expression in H460 cells detected by Western blot. In MIF siRNA-transfected H460 cells (A), we observed a an approximately two (siRNA 1) to five (siRNA2) fold weaker signal of MIF protein expression as compared with the negative control (NC) group normalized to the expression of β-actin (B).

Figure 4

Effect of MIF siRNA on the proliferation of H460 cells. (A); MTT assay showed that treatment of H460 cells with MIF siRNA inhibited their proliferation. Each point in the curve represents the arithmetic mean OD values ± SD from representative experiments that were performed in triplicate. (B); Plate cloning assay indicated that MIF siRNA inhibited the proliferation of H460 cells. Image (C); Quantification of the data obtained from the plate cloning experiments. Data were expressed as arithmetic mean ± SD, Indicated levels of statistical significance were; *: P < 0.05 as compared with the NC Group. NS: Negative control group.

Figure 5

Effect of MIF siRNA on the cell cycle of H460 cells. In propidium iodide stained cells, the cell cycle phases were determined in H460 cells by flow cytometry. The data is described for the cell cycle of H460 cells in the NC group (A), following treatment with MIF siRNA1 (B), and after treatment with MIF siRNA2 (C).

Figure 6

Flow cytometric determination of the effect of MIF siRNA on Apoptosis of H460 cells. (A); Cultured H460 cells were divided into three groups: A: cells transfected with NC siRNA, (B) and (C); cells transfected with MIF siRNA (siRNA1 or siRNA2 respectively). After a 48 h treatment, the cells were harvested for quantitation of apoptosis by determining changes in the cell surface expression of Annexin-V. (B) shows a description of the observed frequency of cells undergoing apoptosis which were found to be much higher in the MIF siRNA1 (18.09 ± 0.41%) and MIF siRNA2 (23.38 ± 2.67%) treated groups than in the negative control (NC) treated group (5.87 ± 1.05 and p < 0.05) of H460 cells respectively.

Figure 7

Effects of MIF siRNA on cellular expression of caspase-3, caspase-4, caspase-8 and Akt in H460 cells. To determine the expression of caspase-3, caspase-4, caspase-8 (A) and Akt (B) in differentially siRNA-treated H460 cells, cultures were harvested after a 48 h treatment, and protein expression patterns assessed by Western immunoblotting analysis and ECL with an appropriate dilution of specific primary antibodies. Beta-actin was used as loading control for all proteins studied.