**4. Discussion**

**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

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.

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 compari-

son to the untreated control group which showed intact DNA.

the necrotic stage are shown.

**3.4. DNA fragmentation analysis**

28 Current Understanding of Apoptosis - Programmed Cell Death

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.

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*. It was also reported that the prenylation together with the 41 , 51 vicinal diol groups in Artonin E had enhanced its affinity to the human estrogen receptor α. This affinity of Artonin E to the human estrogen receptor can be said to be mostly responsible for its better growth inhibition in MCF-7 cells observed in this study. Consistent with our findings, Obiorah et al. [13] discovered that ERα was exclusively responsible for the apoptosis induction of genistein, equol, and coumestrol, compounds which are structurally similar to Artonin E. They confirmed the phenomenon by a gene knockdown of ERα which prevented growth inhibition and apoptosis induced by these phytoestrogens. These support the involvement of the estrogen receptor in the growth inhibitory potential of Artonin E. Similarly, Turner et al. [14] also reported that prenylflavones show selectivity to estrogen receptors. Thus, it is of no doubt, that the affinity of Artonin E for the estrogen receptor is a possible basis for the observed sensitivity of MCF-7 breast cancer cells to the compound.

**Acknowledgements**

**Conflict of interest**

**Author details**

Imaobong Etti<sup>1</sup>

**References**

646-674

1625-1637

513-519

Therapy. 2005;**4**:139-163

The authors declare no conflict of interest.

\*, Rasedee Abdullah<sup>2</sup>

University Putra Malaysia, Serdang, Malaysia

Putra Malaysia, Serdang, Malaysia

\*Address all correspondence to: ettiimaobong@gmail.com

We express our gratitude to the Universiti Putra Malaysia and all the staff of MAKNA-CANCER laboratory, UPM for their immense support in the success of this research. This study was co-supported by TETFund Nigeria, University Putra Malaysia and Ministry of

Apoptosis-Inducing Effect of Artonin E in Breast Cancer http://dx.doi.org/10.5772/intechopen.79205 31

and Arifah Kadir<sup>3</sup>

2 Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine,

3 Department of Veterinary Preclinical Science, Faculty of Veterinary Medicine, Universiti

[1] Hanahan D, Weinberg RA. Hallmarks of cancer: The next generation. Cell. 2011;**144**(5):

[3] Guicciardi M, Gores G. Life and death by death receptors. FASEB Journal. 2009;**23**:

[5] Kroemer G, Reed JC. Mitochondrial control of cell death. Nature Medicine. 2000;**6**(5):

[6] Jin Z, El-Deiry WS. Overview of cell death signaling pathways. Cancer Biology and

[7] Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. Journal of Immunological Methods. 1983;**65**:55-63

[2] Li J, Yuan J. Caspases in apoptosis and beyond. Oncogene. 2008;**27**:6194-6206

[4] Hengartner M. The biochemistry of apoptosis. Nature. 2000;**407**:770-776

1 Department of Pharmacology and Toxicology, University of Uyo, Uyo, Nigeria

Science, Technology and Innovation Malaysia (Vote No. 5450742).

Various modes of cell death include apoptosis, necrosis and autophagy. However, from the results of this study, Artonin E provoked morphological features typical of apoptosis in the Artonin E treated breast cancer cells. In support of this, Carou et al. [15] and Gerl and Vaux [16], reported that apoptosis results in unique morphological changes including cell shrinkage, membrane alteration, DNA fragmentation and nuclear condensation. These features were observed in this study after treating the breast cancer cells with Artonin E. Succinctly, agents that trigger apoptosis are very essential in the management of cancer, giving that a unique hallmark of cancer cells is apoptosis evasion which is also implicated in its pathogenesis [17]. Hence, induction of apoptosis becomes a strategy for cancer drug discovery [18].

A compromise in the phospholipid membrane asymmetry of a cell, results in the externalization of phosphatidylserine. To further strengthen the assessment of the apoptotic mode of cell death, annexin V FITC in combination with a DNA binding fluorochrome, PI were utilized [9, 19, 20]. In this study, Artonin E has been seen to significantly reduce the population of viable breast cancer cells in a concentration and time dependent manner while increasing the population undergoing apoptosis. Thus, treatment of breast cancer cells with Artonin E distorted the integrity of the lipid bilayer of the cancer cells, exposing their phospholipid as detected in this study. These observations qualifies apoptosis as the mode of cell death induced by Artonin E.

Apoptotic endonucleases in the course of apoptosis, degrades chromosomal DNA into fragments [21]. This fragmented DNA can be visualized in a gel electrophoresis. In this study, after treating the cancer cells with Artonin E, its DNA was seen to have degraded into fragments in comparison to the untreated control. This DNA fragment induction by Artonin E, confirms apoptosis as the mode of cell death [22] which was also seen in the morphology of Artonin E treated breast cancer cells and the Annexin V-FITC flowcytometric assays.
