Real-time Imaging of Apoptosis Induction of Human Breast Cancer Cells by the Traditional Chinese Medicinal Herb Tubeimu

Results and Discussion

Induction of apoptosis of MDA-MA-231 RFP-GFP human breast cancer cells by tubeimu in 2D culture. Apoptosis was observed in the tubeimu-treated MDA-MD-231 RFP-GFP cells growing on plastic dishes in 2D culture by nuclear size changes and progressive nuclear fragmentation using confocal imaging (Figure 1). The MDA-MB-231 RFP-GFP cells displayed obvious apoptotic morphological changes after being treated with tubeimu for 24 h. Nuclear size changes, chromatin condensation and progressive nuclear fragmentation were observed (Figure 1). The induction of apoptosis by tubeimu in MDA-MB-231 RFP-GFP cells in 2D culture was dose- and time-dependent, as observed after treatment with 15 μg/ml, 20 μg/ml, and 40 μg/ml tubeimu for 24, 48, and 72 h (Figure 2A, B). The decrease in cell number was also dose- and time-dependent (p<0.01) (Figure 2A). Tubeimu treatment at 40 μg/ml resulted in irregular morphology of the cells and the cell number decreased sharply (Figure 2A). Almost no live cells remained after 48 h treatment with 40 μg/ml tubeimu (Figure 2A). By 72 h, there were almost no live cells remaining even after treatment with 20 μg/ml tubeimu (Figure 2A).

Induction of apoptosis of MDA-MB-231 RFP-GFP human breast cancer cells by tubemu in 3D culture. In 3D Gelfoam® culture, the MDA-MB-231 RFP-GFP cells were more resistant to tubeimu when tested at 20 μg/ml and 25 μg/ml for 24, 48, and 72 h (Figure 3). In 2D culture, the difference between the three different tubeimu dosages and the three different timepoints were more obvious than in 3D culture (Figures 2A, 3 and 4). In 3D Gelfoam® culture, cancer cells assume their natural shape (more in vivo-like) (15). In addition, the cells in 3D Gelfoam® culture attach more tightly to each other and have a broader contact area with each other than that in 2D culture, which may protect the cells from entering apoptosis. However, in the control group in 3D culture at all time points, the cell number increased, in contrast with the number of tubeimu-treated cells which decreased in number over this time period (p<0.01) (Figures 3 and 4). In 3D Gelfoam® culture, the cell morphology changed in the treated cells compared with that of the control group (Figure 3).

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Figure 4.

Comparison of apoptosis in MDA-MB-231 RFP-GFP cells induced by tubeimu in 2D and 3D cultures. MDA-MB-231 RFP-GFP cells were cultured in 2D on plastic dishes and 3D on Gelfoam® in RPMI-1640 medium. Please see Figures 2 and 3 for details. The frequency of apoptosis in treated and control cultures was compared ([pentastar2][pentastar2]=p<0.01).

Induction of DNA fragmentation in MDA-MD-231 RFP-GFP cells by tubeimu. Agarose-gel electrophoresis detected DNA fragmentation in the MDA-MB-231 RFP-GFP cells after 48 h treatment with tubeimu in 2D culture, with an increase at 72 h. In contrast, morphological changes observed by fluorescence microscopy revealed apoptosis induction by tubeimu by 24 h. Fluoresence microscopy of cancer cells labeled with GFP in the nucleus and RFP in the cytoplasm is therefore more sensitive than DNA electrophoresis for detecting apoptosis (Figure 5).

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Figure 5.

Fragmentation of MDA-MB-231 RFP-GFP cells after tubeimu treatment. Lanes contain DNA from the following cultures: (1) tubeimu at 15 μg/ml for 72 hours in 2D culture, (2) tubeimu at 15 μg/ml for 48 hours, (3) tubeimu at 15 μg/ml for 24 hours, (4) untreated control, (5) DNA marker (100bp), (6) DNA marker (smallest size is 1,000 bp).

TCM is a vast potential resource for cancer treatment. However, in order to determine which herbs or their combination have anticancer activity that can be used widely, modern methods must be used to evaluate them. In the present report, we utilized human breast cancer cells engineered to express RFP in the cytoplasm and GFP in the nucleus (9-11). These cells clearly report nuclear-cytoplamic dynamics in real time and are ideal to reveal the onset and progression of treatment-induced apoptosis. The findings of this report, demonstrate the potential of tubeimu for cancer therapy.