**3. The subpopulation in the MDA-MB-231 cells**

Most of the cancer cell lines have recently been demonstrated by flow cytometry to contain a subpopulation of CD44+/CD24- where the MDA-MB-231 cells are found to contain a high percentage of the CD44+/CD24- subpopulation (85±5%) in the cells (Sheridan *et al*., 2006). Other cell lines that contain a high level of this subpopulation are MDA-MB-436 (72±5%), Hs578T (86±5%) and SUM1315 (97±3%) (Table 1). The subpopulation is shown to possess the capacity for self-renewal and the generation of heterogeneous progeny in the cells. Moreover, the subpopulation of the breast cancer cells has been reported to have stem/progenitor cell properties that contribute a unique ability to allow these cells to invade. Similar to the ability of the MSCs that was described above, the inherent properties of this subpopulation may impart their transformed counterparts with the ability to evade traditional antitumour therapies and to establish breast cancer metastasis (Reya *et al*., 2001; Behbod *et al*., 2005; Dean *et al*., 2005). Several studies suggested that this subpopulation of cells, as a subset of human breast cancer cells, possessed an enhanced ability to form tumour in immunocompromised mice (Al-Hajj *et al*., 2003; Ponti *et al*., 2005). However, the potential of this subpopulation to establish breast cancer metastasis in the cell line remains unclear.

The expression levels of pro-invasive genes, such as interleukin-1-alpha (IL-1), IL-6, interleukin-8 (IL-8) and urokinase plasminogen activator (UPA), are higher in the cell lines that contained a significant CD44+/CD24- subpopulation (Sheridan *et al*., 2006). The results indicate that the cell lines with a significant number of CD44+/CD24 subpopulation are more invasive is consistent with the studies that demonstrate the metastatic process in breast cancer cells requires the following: (1) ECM degradation-associated proteins, including the

The Mesenchymal-Like Phenotype of the MDA-MB-231 Cell Line 395

Before the subpopulation and invasiveness of the MDA-MB-231 cells can be further elucidated, hierarchy of the breast cancer stem cells in the breast cancer cell compartment should be understood. A breast cancer stem cell, as described in the cancer stem cell compartment hierarchy, is capable of undergoing an asymmetric cell division to generate one cell that is identical to itself (orange colour) and one that it is more committed towards a certain differentiation pattern (a breast cancer cell, grey colour) (Cariati & Purushotham, 2008; Fig. 7).

Fig. 7. A breast cancer stem cells-breast cancer cells compartment hierarchy. A breast cancer stem cell is capable of going through an asymmetric cell division to generate one cell that is identical to itself (orange colour) and one that tends toward a certain differentiation pattern

The formation of the identical cell ensures that the cancer stem cell compartment is maintained throughout its time in the subpopulation. These distinct cells undergo series of divisions and differentiation steps that result in the generation of a terminally differentiated

(breast cancer cells, grey colour) (Modified from Cariati & Rurushotham, 2008).

**4. Mesenchymal-like phenotype of the MDA-MB-231 cells** 

UPA/UPA receptor system and MMPs; (2) cytokines, including interleukin-1 (IL-1), IL-6, IL-8, interleukin-11 (IL-11), tumour necrosis factor (TNF) and transforming growth factor-beta 1 (TGF-1); and (3) chemokines and their receptors, including stromal cell-derived factor-1 alpha (SDF-1) and CXC chemokine receptor (CXCR4) (Edwards and Murphy, 1998; Dumont and Arteaga, 2003; Kang *et al*., 2003; Yodkeeree *et al*., 2010). In addition, a recent study described the role of nuclear factor-kappa B (NF-B) and its ligand in the metastasis of breast cancer cells to the bone matrix (Jones *et al*., 2006). All of these factors may be directly related to breast cancer metastasis. However, the contribution of the subpopulation of CD44+/CD24 to the pro-invasive factors in breast cancer cells remains unclear.


Table 1. Subpopulation of CD44+/CD24- in commonly used breast cancer cell lines. The CD44 and CD24 expression patterns in the subpopulation CD44+/CD24- were determined by flow cytometry. CD44 and CD24 were detected by a combination of fluorochromeconjugated monoclonal antibodies against human CD44 (FITC) and CD24 (PE), respectively (Sheridan *et al*., 2006).

Demethoxycurcumin (DMC) is recently demonstrated to inhibit the adhesion, migration and invasion of the MDA-MB-231 cells (Yodkeeree *et al*., 2010). According to the study, the DMC-treated MDA-MB-231 cells contained decreasing levels of ECM degradationassociated proteins, which included MMP-9, membrane type-1 MMP (MT1-MMP), UPA and UPAR. DMC reduced also the expression of intercellular adhesion molecule-1 (ICAM-1) and CXCR4 in the MDA-MB-231 cells. These molecules are involved in the modulation of the tumour metastasis process. In addition, the study showed that treatment of the MDA-MB-231 cells with DMC inhibited the DNA binding activity of NF-B, which is known to mediate the expression of MMPs, UPA, UPAR, ICAM-1 and CXCR4 in breast cancer cells. These results indicated also that NF-B may play a role in the invasion process of the MDA-MB-231 cells. All of these findings suggest the presence of a correlation between the above molecules and the invasiveness of the MDA-MB-231 cells. However, the specific correlation between the above molecules and the subpopulation in the MDA-MB-231 cells has yet to be fully elucidated. The inhibition or depletion of the progenitor factor from the subpopulation is hypothesised to reduce the expression of the above molecules, thereby reducing the invasiveness of the MDA-MB-231 cells. Therefore, it is essential to identify more surface markers that can specifically be used to isolate the subpopulation. By targeting this subpopulation in the cells, the expression of the above molecules and the invasiveness of the MDA-MB-231 cells can be further elucidated.

UPA/UPA receptor system and MMPs; (2) cytokines, including interleukin-1 (IL-1), IL-6, IL-8, interleukin-11 (IL-11), tumour necrosis factor (TNF) and transforming growth factor-beta 1 (TGF-1); and (3) chemokines and their receptors, including stromal cell-derived factor-1 alpha (SDF-1) and CXC chemokine receptor (CXCR4) (Edwards and Murphy, 1998; Dumont and Arteaga, 2003; Kang *et al*., 2003; Yodkeeree *et al*., 2010). In addition, a recent study described the role of nuclear factor-kappa B (NF-B) and its ligand in the metastasis of breast cancer cells to the bone matrix (Jones *et al*., 2006). All of these factors may be directly related to breast cancer metastasis. However, the contribution of the subpopulation of CD44+/CD24-

**No. Cell Line CD44+/CD24- Cell Type Classification**  a MDA-MB-231 85±5 Mesenchymal b MDA-MB-436 72±5 Myoepithelial c Hs578T 86±5 Mesenchymal d MDA-MB-468 3±1 Basal e MCF-7 0 Luminal f T47-D 0 Luminal g ZR-75-1 0 Luminal h BT-474 0 Luminal/ErbB2+ i SK-BR-3 0 Luminal/ErbB2+ j MCF-10A 17±4 Basal

Table 1. Subpopulation of CD44+/CD24- in commonly used breast cancer cell lines. The CD44 and CD24 expression patterns in the subpopulation CD44+/CD24- were determined by flow cytometry. CD44 and CD24 were detected by a combination of fluorochromeconjugated monoclonal antibodies against human CD44 (FITC) and CD24 (PE), respectively

Demethoxycurcumin (DMC) is recently demonstrated to inhibit the adhesion, migration and invasion of the MDA-MB-231 cells (Yodkeeree *et al*., 2010). According to the study, the DMC-treated MDA-MB-231 cells contained decreasing levels of ECM degradationassociated proteins, which included MMP-9, membrane type-1 MMP (MT1-MMP), UPA and UPAR. DMC reduced also the expression of intercellular adhesion molecule-1 (ICAM-1) and CXCR4 in the MDA-MB-231 cells. These molecules are involved in the modulation of the tumour metastasis process. In addition, the study showed that treatment of the MDA-MB-231 cells with DMC inhibited the DNA binding activity of NF-B, which is known to mediate the expression of MMPs, UPA, UPAR, ICAM-1 and CXCR4 in breast cancer cells. These results indicated also that NF-B may play a role in the invasion process of the MDA-MB-231 cells. All of these findings suggest the presence of a correlation between the above molecules and the invasiveness of the MDA-MB-231 cells. However, the specific correlation between the above molecules and the subpopulation in the MDA-MB-231 cells has yet to be fully elucidated. The inhibition or depletion of the progenitor factor from the subpopulation is hypothesised to reduce the expression of the above molecules, thereby reducing the invasiveness of the MDA-MB-231 cells. Therefore, it is essential to identify more surface markers that can specifically be used to isolate the subpopulation. By targeting this subpopulation in the cells, the expression of the above molecules and the invasiveness of the

the pro-invasive factors in breast cancer cells remains unclear.

(Sheridan *et al*., 2006).

MDA-MB-231 cells can be further elucidated.

to
