Cell-Based Assays in Cancer Research

*Biba Vikas and Sukumaran Anil*

## **Abstract**

Cell growth is referred to as cell proliferation, that is, the increase in cell numbers during repeated cell division. Cell growth can be defined as the enlargement of cell volume, which might take place in the absence of cell division. Growth and reproduction are features of cells in all living organisms. All cells reproduce by dividing into two, with each parental cell giving rise to two daughter cells each time they divide. Various genes are involved in the control of cell division and growth. Reproduction in unicellular organisms are referred to cell division and in multicellular organisms it is tissue growth and maintenance. Survival of the eukaryotes depends upon interactions between various cell types, that helps in the balanced distribution. This is achieved through the highly regulated process of cell proliferation. Knowledge in cell cycle is necessary to determine the best time to collect cells, to harvest cell products, or to move cells to a new growth environment. Cancer cells do not die at the natural point in a cell's life cycle. Cancer cells occur as the results of cellular changes caused by the uncontrolled growth and division of cells. The chapter focuses on cancer cell maintenance, apoptosis, and its detection assays.

**Keywords:** cancer cell maintenance, apoptosis, cytotoxicity, cell-based assays

#### **1. Introduction**

Cell division and the increase in cell quantity is called cell growth. Cell growth happens in favorable nutrient conditions. It is the process by which cells accumulate mass and increase in physical size. The growth of tissues that are not self-renewing occurs by a combination of increase in number and increase in size of the component cells. Cells will progress unimpeded through the cell cycle and divide; one cell will become two, two will become four, four will become eight, and so on. Cellular growth is ensured by the alternation of DNA duplication and cell division cycles [1]. When the cell reaches maximum size, the important point is that the surface area to the volume ratio becomes smaller as the cell gets larger. When the cell grows beyond a certain limit, inadequate material will be able to cross the membrane sufficient to accommodate the increased cellular volume. Increase of size and change in shape of a developing organism depend on the increase in the number and size of cells. The increase in cell number is due to cellular reproductive mechanism called mitosis. Cells are limited to their programmed size because the cell membrane must transport oxygen and food into the cell, as well as transport waste like CO2 and H2O out of the cell. As the cell grows, the inside grows faster than the outside. Cellular growth is ensured by the alternation of DNA duplication and cell division cycles. As the cell grows, the inside grows faster than the outside. Cellular growth is ensured

by the alternation of DNA duplication and cell division cycles; through transcription the coupling of cell divisions is taking place in metabolic pathways [2].

*Cancer cell growth*: cancer is a condition where cells grow uncontrollably in a specific part of the body. Cancerous cells can invade and destroy surrounding healthy tissue and organs. Cancer cells divide relentlessly, forming solid tumors or flooding the blood with abnormal cells. Cell division is a normal process for growth and repair. A parent cell divides to form two daughter cells and is used to build new tissue, or it is used to replace cells that have died because of aging or damage. Healthy cells stop dividing when there is no longer a need for more daughter cells, but cancer cells continue the divisions. They are capable of spreading from one part of the body to another in a process known as metastasis [3]. Cell proliferation and cell death are such diametrically opposed cellular fates that are linked and interdependent processes [4, 5].

Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a suitable artificial environment. In vitro assays are performed to check the proliferation that reflects cellular responses to various stimuli. These techniques help to observe cell division and quantity of cells. Cell culture technology shows a good progress in biology and is heavily dependent on cell culture technology. Chemotherapeutic agent phenoxodiol, a synthetic analog of daidzein, a well-known isoflavone from soybean (*Glycine max*) was developed as a therapeutic agent against cervical, ovarian, prostate, renal, and vaginal cancers. They induce apoptosis through the inhibition of antiapoptotic proteins [6]. Apoptotic shrinkage, disassembly into apoptotic bodies, and engulfment of individual cells characteristically occur without associated inflammation. This could then be the consequence of releasing intracellular contents into tissues. This could be the consequence of releasing intracellular contents into tissues; the mitochondria remain unchanged morphologically [7].

#### **1.1 The intrinsic pathway for programmed cell death**

The signaling pathway for programmed cell death involves non-receptormediated intracellular signals inducing activities in the mitochondria that initiate apoptosis. Stimuli for the intrinsic pathway are caused by viral infections or damage to the cell by toxins, free radicals, or radiation. Damage to the cellular DNA can also induce the activation of the intrinsic pathway for programmed cell death [8]. Proapoptotic proteins activate caspases to mediate the destruction of the cell through different pathways. These proteins translocate to the nucleus of cells, thereby inducing DNA fragmentation which is a hallmark of apoptosis. The members of the Bcl-2 family of proteins and the tumor suppressor protein p53 regulate proapoptotic event in the mitochondria (**Figure 1a**). The Bcl-2 family members of proteins may be pro- or antiapoptotic. Bcl-2, Bcl-x, Bcl-xL, Bcl-XS, Bcl-w, and BAG are the antiapoptotic proteins. These proteins are currently under investigation as potential targets for anticancer therapy. Bcl-10, Bax, Bak, Bid, Bad, Bim, Bik, and Blk are proapoptotic proteins. The upregulation of these proteins or their increased activation may offer an approach for cancer therapy [9]. Cellular pathways that modulate the activities of the p53 protein are also currently being evaluated as targets for potential anticancer therapies [10].

#### **1.2 The extrinsic pathway for programmed cell death**

The extrinsic signaling pathway leads to apoptosis that is through transmembrane death receptors, members of the tumor necrosis factor (TNF) receptor gene super family. Members of this receptor family bind to extrinsic ligands and

**103**

**Figure 1.**

*Cell-Based Assays in Cancer Research*

*DOI: http://dx.doi.org/10.5772/intechopen.90226*

transduce intracellular signals that result in the destruction of the cell [11]. The most well-characterized ligands of these receptors are FasL, TNF-alpha, Apo3L, and Apo2L, and its corresponding receptors are FasR, TNFR1, DR3, and DR4/DR5, respectively [8, 9, 12]. Molecules that stimulate the activity of these proapoptotic proteins or activate these receptors are currently under the evaluation for their therapeutic potential in the treatment of cancer, including hematologic malignancies. The signal transduction of extrinsic pathway involves various caspases which are proteases with specific cellular targets. Once activated, the caspases affect several cellular functions as part of a process that results in the death of the cells [8]. This visible transformation of apoptosis is accompanied by biochemical changes. Those at the cell surface include the externalization of phosphatidyl serine and other alterations that promote recognition by phagocytes. Intracellular changes include the degradation of the chromosomes of the chromosomal DNA into high molecular weight and oligonucleosomal fragments and cleavage of a specific subset of cellular polypeptide [13, 14]. These cleavages are accompanied by a family of

*(a) The intrinsic pathway for programmed cell death. (b) The extrinsic pathway for programmed cell death.*

Cell culture is used in vitro in cell biology, tissue morphology, and mechanisms of diseases, drug action, protein production, and the development of tissue engineering. The stage of the culture after the cells can be isolated from the tissue and proliferates under favorable conditions until they reach confluency. In this stage, the cells have to be passaged or subcultured by transferring them to a new vessel with fresh growth medium to provide more room for continued growth. Various researches in cancer biology are based on experiments using two-dimensional cell culture by growing cells in flat dishes, made of plastic. The dish culture system is mainly used for developing adherent two-dimensional cell monolayers. 3D cell culture applications are usually beneficial in tissue engineering and regenerative

intracellular proteases called caspases (**Figure 1b**).

**2. 2D and 3D cell culture systems**

#### *Cell-Based Assays in Cancer Research DOI: http://dx.doi.org/10.5772/intechopen.90226*

*Cell Growth*

pendent processes [4, 5].

morphologically [7].

**1.1 The intrinsic pathway for programmed cell death**

targets for potential anticancer therapies [10].

**1.2 The extrinsic pathway for programmed cell death**

by the alternation of DNA duplication and cell division cycles; through transcription the coupling of cell divisions is taking place in metabolic pathways [2]. *Cancer cell growth*: cancer is a condition where cells grow uncontrollably in a specific part of the body. Cancerous cells can invade and destroy surrounding healthy tissue and organs. Cancer cells divide relentlessly, forming solid tumors or flooding the blood with abnormal cells. Cell division is a normal process for growth and repair. A parent cell divides to form two daughter cells and is used to build new tissue, or it is used to replace cells that have died because of aging or damage. Healthy cells stop dividing when there is no longer a need for more daughter cells, but cancer cells continue the divisions. They are capable of spreading from one part of the body to another in a process known as metastasis [3]. Cell proliferation and cell death are such diametrically opposed cellular fates that are linked and interde-

Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a suitable artificial environment. In vitro assays are performed to check the proliferation that reflects cellular responses to various stimuli. These techniques help to observe cell division and quantity of cells. Cell culture technology shows a good progress in biology and is heavily dependent on cell culture technology. Chemotherapeutic agent phenoxodiol, a synthetic analog of daidzein, a well-known isoflavone from soybean (*Glycine max*) was developed as a therapeutic agent against cervical, ovarian, prostate, renal, and vaginal cancers. They induce apoptosis through the inhibition of antiapoptotic proteins [6]. Apoptotic shrinkage, disassembly into apoptotic bodies, and engulfment of individual cells characteristically occur without associated inflammation. This could then be the consequence of releasing intracellular contents into tissues. This could be the consequence of releasing intracellular contents into tissues; the mitochondria remain unchanged

The signaling pathway for programmed cell death involves non-receptormediated intracellular signals inducing activities in the mitochondria that initiate apoptosis. Stimuli for the intrinsic pathway are caused by viral infections or damage to the cell by toxins, free radicals, or radiation. Damage to the cellular DNA can also induce the activation of the intrinsic pathway for programmed cell death [8]. Proapoptotic proteins activate caspases to mediate the destruction of the cell through different pathways. These proteins translocate to the nucleus of cells, thereby inducing DNA fragmentation which is a hallmark of apoptosis. The members of the Bcl-2 family of proteins and the tumor suppressor protein p53 regulate proapoptotic event in the mitochondria (**Figure 1a**). The Bcl-2 family members of proteins may be pro- or antiapoptotic. Bcl-2, Bcl-x, Bcl-xL, Bcl-XS, Bcl-w, and BAG are the antiapoptotic proteins. These proteins are currently under investigation as potential targets for anticancer therapy. Bcl-10, Bax, Bak, Bid, Bad, Bim, Bik, and Blk are proapoptotic proteins. The upregulation of these proteins or their increased activation may offer an approach for cancer therapy [9]. Cellular pathways that modulate the activities of the p53 protein are also currently being evaluated as

The extrinsic signaling pathway leads to apoptosis that is through transmembrane death receptors, members of the tumor necrosis factor (TNF) receptor gene super family. Members of this receptor family bind to extrinsic ligands and

**102**

transduce intracellular signals that result in the destruction of the cell [11]. The most well-characterized ligands of these receptors are FasL, TNF-alpha, Apo3L, and Apo2L, and its corresponding receptors are FasR, TNFR1, DR3, and DR4/DR5, respectively [8, 9, 12]. Molecules that stimulate the activity of these proapoptotic proteins or activate these receptors are currently under the evaluation for their therapeutic potential in the treatment of cancer, including hematologic malignancies. The signal transduction of extrinsic pathway involves various caspases which are proteases with specific cellular targets. Once activated, the caspases affect several cellular functions as part of a process that results in the death of the cells [8]. This visible transformation of apoptosis is accompanied by biochemical changes. Those at the cell surface include the externalization of phosphatidyl serine and other alterations that promote recognition by phagocytes. Intracellular changes include the degradation of the chromosomes of the chromosomal DNA into high molecular weight and oligonucleosomal fragments and cleavage of a specific subset of cellular polypeptide [13, 14]. These cleavages are accompanied by a family of intracellular proteases called caspases (**Figure 1b**).
