**3.3. Annexin V-FITC assay flowcytometric analysis**

The externalized phosphatidyl serine was detected with Annexin V-FITC dye as an indication of apoptosis induced upon treatment of breast cancer cells with Artonin E. From the results, there was a significant (p < 0.05) shift in the population of the breast cancer cells from viability

extracted DNA was assessed with a nanodrop spectrophotometer (nanodrop lite spectrophotometer, Thermo Scientific, USA). The DNA sample was mixed with 1× loading dye and electrophoresed on 1% agarose gel 75 V for 1 h and stained with GelRed™ nucleic acid gel stain. The fragmented DNA was visualized under UV transilluminator and photographed with a chemiluminescence image analyzer system (Chemi-Smart, Vilber Lourmat, Germany).

All data collected were analyzed using the GraphPad Prism 5.0 (GraphPad Software Inc., La Jolla, CA, USA). One way analyses of variance was performed, followed by Turkey's *post ho*c tests to compare replicate means of treatment and control groups. The significance was set at

Artonin E, at concentrations of 6.9, 5.10 and 3.77 μM induced a half maximal growth inhibitory effect on estrogen receptor-positive breast cancer cells (**Figure 1**) at 24, 48 and 72 h

The results of the AO and PI double staining analysis showed that Artonin E treated breast cancer cells displayed morphological features typical of apoptosis. These features included chromatin condensation and membrane blebbing. The control breast cancer cells which were not exposed to Artonin E had their normal nuclear structure displayed as green fluorescence. Early apoptotic cells were observed as bright green fluorescence resulting from the interposition of acridine orange with the fragmented DNA whereas late stage apoptosis (**Figure** 2**C** and **D**) was observed as reddish-orange, resulting from the binding of propidium iodine to denatured DNA. The apoptosis inducing effect of Artonin E was observed to be time and

Challenging the cancer cells with Artonin E caused a significant (p < 0.05) loss in the proportion of viable MCF-7 breast cancer cells (**Figure 3**) from 94.5% to 67, 38, and 21.5% after treatment with 3, 10, and 30 μM Artonin E respectively. There was no significant (p ≥ 0.05) increase in necrotic breast cancer cells except after exposure to 30 μM of Artonin E. This percentage decreased. There was a concentration and time dependent significant (p < 0.05) increase in the

The externalized phosphatidyl serine was detected with Annexin V-FITC dye as an indication of apoptosis induced upon treatment of breast cancer cells with Artonin E. From the results, there was a significant (p < 0.05) shift in the population of the breast cancer cells from viability

**3.2. Artonin E treated MCF-7 breast cancer cells displayed morphology of apoptosis**

**3.1. Growth inhibitory effect of Artonin E on MCF-7 breast cancer cells**

**2.9. Statistical analysis**

26 Current Understanding of Apoptosis - Programmed Cell Death

p < 0.05.

**3. Results**

respectively.

concentration dependent.

proportion of cells that entered the apoptotic phase.

**3.3. Annexin V-FITC assay flowcytometric analysis**

**Figure 2.** Acridine orange/propidium iodide double staining of MCF-7 cells after 24 h exposure. (A) Control, (B) 3, (C) 10, (D) 30 μM Artonin E. VC = viable cells; BL = cell membrane blebbing; CC = chromatin condensation; EA = early apoptosis; LA = late apoptosis; MN arginated nuclear chromatin; SN secondary necrosis. Magnification: 200×.

**Figure 3.** Quantification of early and late apoptotic MCF-7 cells after AO/PI double staining analysis. All values are mean ± standard deviation. \*at each time point, means significantly different for control at p < 0.05.

**Figure 4.** Representative histogram analysis of the Annexin V assay in MCF-7 cells treatment with Artonin E at 24 and 48 h. Viable cells population are shown in the lower left quadrant, while those undergoing early apoptosis are in the lower right quadrant. The upper right quadrant shows cells at late stage of apoptosis, and in upper left corner, cells at the necrotic stage are shown.

**4. Discussion**

with 4 μg/mL camptothecin, the DNA marker is a 1 kb ladder.

The death of cancer cells in a tumor is the ultimate goal of cancer drug discovery. Unlike normal cells, cancer cells possess the ability to proliferate uncontrollably while evading apoptosis. Apoptosis induction is thus a valuable characteristic of a potential anticancer drug. Hence, the cytotoxic effect of a compound can be accessed by investigating its growth inhibition on the target cancer cells. It is also vital to examine the mode of cell death induced by the compound. In this study, Artonin E, previously shown to be drug-like with a better *in silico* growth inhibitory properties when compared with similar structural analogues [11], was evaluated for its *in vitro* growth inhibition and mode of cell death induced in MCF-7 breast cancer cell line.

**Figure 5.** DNA fragmentation in MCF-7 cells treated with Artonin E after 24 h. The positive control cells were treated

Apoptosis-Inducing Effect of Artonin E in Breast Cancer http://dx.doi.org/10.5772/intechopen.79205 29

Artonin E was found to significantly inhibit the proliferation of this breast cancer cells in a dose and time dependent manner. At 24, 48 and 72 h, Artonin E showed half maximal inhibitory concentrations of 6.90, 5.10 and 3.77 μM, respectively. MCF-7 is estrogen receptor positive, progesterone positive and HER2 (human epidermal growth factor 2) positive and has wild type p53 [12]. Etti et al. [11] reported the *in silico* affinity of Artonin E to the human estrogen receptor α and pin pointed Artonin E as having greater binding affinity for the estrogen receptor α among the reported structural analogues from the *Artocarpus*.

to late apoptosis (**Figure 4**). This effect was concentration as well as time dependent. After 24 h exposure of the cancer cells to Artonin E, the percentage viability decreased from 97.78% to 81.27, 36.33 and 12.0 6% when treated with 3, 10 and 30 μM of Artonin E respectively.

#### **3.4. DNA fragmentation analysis**

Another unique feature of apoptosis is DNA fragmentation which can be visualized by agarose gel electrophoresis [10]. From the results, nuclear DNA fragmentation occurred after treatment with Artonin E. This was observed in the breast cancer cells (**Figure 5**) in comparison to the untreated control group which showed intact DNA.

**Figure 5.** DNA fragmentation in MCF-7 cells treated with Artonin E after 24 h. The positive control cells were treated with 4 μg/mL camptothecin, the DNA marker is a 1 kb ladder.
