**1. Introduction**

Breast cancers, like other forms of cancer, possess the ability for uncontrolled proliferation while resisting cell death [1]. This undue proliferation is a consequence of apoptosis evasion as well as loss of normal cell cycle control. Apoptosis is an efficient and uniquely regulated mode of cell death, involving the interplay of many factors. In mammals, there are two major pathways of apoptosis viz., the death receptor mediated pathway (extrinsic pathway) and

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

the mitochondrial mediated pathway (intrinsic pathway). Caspases are the key regulatory proteins for both pathways [2]. The extrinsic pathway requires the binding of ligands such as tumor necrosis factor α (TNFα), Fas ligand (Fas-L) and TNF-related apoptosis-inducing ligand (TRAIL) to the death receptor on cell surfaces and corresponding transduction of signals leading to apoptosis [3]. The intrinsic pathway, on the other hand, is mediated by the release of cytochrome c by the mitochondrial following DNA damage. The released cytochrome c then induces the formation of apoptosomes composed of apoptotic protease activating factor 1 (Apaf-1), procaspase 9, and either ATP or dATP [4]. Caspase 9 is a downstream factor that activates the executioner caspase-3, which cleaves substrates such as Poly (ADP-ribose) polymerase (PARP) [5]. The activated caspase 3 then initiates the caspase cascade that culminates in the demolition of the cell [6].

**2.2. Cryogenic preservation and recovery**

flask (TPP, Switzerland), incubated at 37°C in a CO<sup>2</sup>

is the initial cell concentration, V<sup>1</sup>

incubated overnight at 37°C to facilitate attachment.

replaced with fresh complete growth medium.

using a hemocytometer. Briefly, 0.5 × 10<sup>5</sup>

final cell concentration, and V<sup>2</sup>

**2.4. Preparation of the test agents**

ate wells in four replicates.

**2.3. Plating**

formula:

where M1

The cells were preserved in liquid nitrogen to avoid loss of their original characteristics. Briefly, the cells growing in the exponential phase were subcultured. Following detachment and centrifugation, the cells were resuspended in freezing medium containing 90% FBS and

Subsequently, 1 mL of cell suspension was transferred into each labeled cryotube and allowed to stand at −20°C for 2 hours and at −80°C overnight before storage in liquid nitrogen (−196°C) (CBS Cryosystem). When needed, the cells were recovered by thawing with gentle agitation in water bath at 37°C for approximately 2 min. The cell suspension was transferred into a sterile 15 mL centrifuge tube (TPP, Switzerland) containing prewarmed complete growth medium in a biosafety cabinet and centrifuged at 1000 rpm (Hettich Universal 32 R centrifuge, DJB Labcare Ltd., UK) for 5 min at room temperature to remove the cryoprotective agent (DMSO).

cells/mL.

23

culture

cells/

is the

incubator. After 2 days, the medium was

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

cells/mL were dispensed into each well of a 96-well

is the initial cell suspension volume, M2

10% DMSO, as a cryoprotective agent, to yield a final cell density of 2 to 5 × 10<sup>6</sup>

Finally, the cell pellets were resuspended in culture media and transferred to a 25cm2

At about 80% confluency, the cells were collected and the concentration was determined

mL, the initial cell concentration was adjusted with culture medium using the following

M1 V<sup>1</sup> = M2 V<sup>2</sup> (1)

is the final cell suspension volume.

Using a multichannel pipette, 100 μL of the cell suspension was dispensed into each well, except the blank wells, which received 200 μL of culture medium. The cell-seeded plate was

Artonin E was kindly donated by Dr. Najihah Hashim of the Department of Pharmacy, Faculty of Medicine, University of Malaya. The compound was isolated from *Artocarpus elasticus*, characterized and identified as reported by [8]. The standard agents, Tamoxifen and Paclitaxel were purchase from Sigma Aldrich, St. Louis, MO, USA. These agents were dissolved in DMSO and normal saline respectively and then diluted with respective medium with highest final DMSO concentration of 0.1% for *in vitro* cell culture studies. They were diluted serially using culture medium to obtain concentrations in the range of 1.56–100 μM. About 100 μL of each concentration of Artonin E, Tamoxifen and Paclitaxel solutions was added into appropri-

flat bottom tissue culture plate. To obtain the desired plating concentration of 0.5 × 10<sup>5</sup>

In this study, the colorimetric microculture tetrazolium assay [7] was employed to access the cells viability while assays including morphology studies, Annexin V-FITC Assay and DNA fragmentation were thereafter utilized to ascertain the mode of the cell death.
