**2. Platinum-based drugs and breast cancer cells**

Platinum-based drugs (PBDs) are used for adjuvant chemotherapy to reduce mortality from breast cancer with reversible side-effects [12]. A key feature of platinum based drugs is that once platinum salts enter cells, they can bind to DNA to form Platinum-DNA adducts that can cause damage to the DNA. Following DNA damage, cell cycle checkpoints are activated to repair either the damaged DNA or induce apoptosis (cell death) [13, 14]. Thus, the ultimate goal in the application of platinum-based chemotherapy is to shift the dynamics away from cell growth and survival in favor of cell differentiation and apoptosis. This will in turn reduce and eliminate tumor progression and malignancy [15].

Although PBDs are initially effective, their efficacy is limited by the occurrence of resistance, which is attributed to alterations in cellular pathways such as DNA repair, drug transport, drug metabolism and apoptosis [16]. Several studies have explored the cellular and molecular pathways involved in the mechanism of PBDs resistance to breast cancer [13, 16–18]. However, only a few ultrastructural studies on the intracellular organelles of breast cancer cells have been performed to determine the effectiveness of these drugs.

#### **2.1. Surface structure of breast cancer cells differ from normal breast cells**

HER2 expression is variable (positive or negative). Luminal B tumors have higher proliferation and poorer prognosis than luminal A tumors. HER2 overexpression group accounts for 15% of all invasive breast cancers and the tumor usually tends to be ER/PR negative. The basal class is typically ER/PR negative and HER2 negative, hence the name TNBC [3]. It comprises about 15% of all invasive breast cancers and have a fairly poor prognosis. Normal-like tumors account for 7.8% of all breast cancer cases in a lymph-node negative cohort. It is positive for

Due to this heterogeneity, the treatment is complicated and the therapeutic strategies should be selected carefully. To overcome the disease, it is imperative that each patient be treated individually according to the morphological classification with molecular parameters and sensitivity to available therapy. Treatment of breast cancer includes surgery, radiation therapy, hormone-modification therapy and chemotherapy (anticancer drugs). Chemotherapy treatment has markedly reduced the risk for recurrence and mortality after primary treatment

One of the major modes of action of chemotherapeutic drugs may be the activation of apoptosis (programmed cell death) [7]. Hence, anticancer drugs are associated with the activation of proapoptotic genes and the suppression of antiapoptotic genes. The attenuation of proapoptotic genes and increases in antiapoptotic genes causes resistance to apoptosis [8]. Hence, in order to increase the therapeutic effect of chemotherapy, there is a need to assess the molecular mechanisms of apoptosis induced anticancer drugs. This may lead to new strategies for

In this chapter, we hope to summarize three attempted molecular biology studies on breast cancer that have contributed to further knowledge in this field. We have compared the effects of platinum based-chemotherapeutic drugs such as cisplatin, carboplatin and oxaliplatin on the ultrastructure of the three human breast cancer cell lines representing the most diagnosed types; MDA-MB-231, MCF-7 and BT-474 [9]. We have particularly demonstrated the role of cisplatin in inducing apoptosis in MDA-MB-231 via the endoplasmic reticulum- mediated calpain-1 pathway [10]. At the same time, we have assessed the expression of calpain-1 as a potential prognostic factor in TNBC tissues [11]. Understanding the pathways by which platinum-based drugs induce apoptosis and how these pathways are altered in chemoresistance can provide valuable information necessary to target specific cell death pathways in the

Platinum-based drugs (PBDs) are used for adjuvant chemotherapy to reduce mortality from breast cancer with reversible side-effects [12]. A key feature of platinum based drugs is that once platinum salts enter cells, they can bind to DNA to form Platinum-DNA adducts that can cause damage to the DNA. Following DNA damage, cell cycle checkpoints are activated to repair either the damaged DNA or induce apoptosis (cell death) [13, 14]. Thus, the ultimate goal in the application of platinum-based chemotherapy is to shift the dynamics away from

of breast cancer and have increased the 5- and 10-year survival rates [6].

the enhancement of the antitumor effect against target organs.

treatment of clinically resistant breast cancer.

**2. Platinum-based drugs and breast cancer cells**

ER and PgR but negative for HER2 [4, 5].

148 Breast Cancer and Surgery

We used SEM to compare the surface morphology between three models of breast cancer cells, each of which is characterized with a distinct immunohistochemical profile. The MCF-7 cell line was used to represent the luminal A breast cancer [19], the BT-474 cell line, the luminal B tumor [20] and the MDA-MB-231 cell line, the basal-like subtype, TNBC [3].

Normal breast cells, MCF-10A, revealed round shape cells characterized by short lamellipodia, whereas, the breast cancer cells had a semiflattened surface structure containing microvilli with extending lamellipodia. Lamellipodia consist of protrusive filamentous actin and signaling proteins, which play a role in cell migration and cell–cell communication. These surface protrusions are important in enhancing movement and adhesion to the surrounding stroma [21]. They appeared to be lesser in number and finer in shape for both MCF-7 and BT-474 cells but higher in number and thicker for MDA-MB-231 cells. Since MDA-MB-231 cells are advanced cancer cells with metastatic characteristics, therefore it is not surprising for these cells to contain higher numbers of lamellipodia on their cell surface. This is indicative of their importance of cell shape modifications in their invasiveness process unlike the normal breast cells. These distinct features of TNBCs *in vivo* models might demonstrate their aggressiveness and give them a metastatic potential [21–23]. TEM micrographs revealed the absence of nuclei in the MDA-MB-231 cells whereas more than one nucleus were detected in MCF-7 and BT-474 cells.

#### **2.2. Effect of PBDs on the cell membrane of breast cancer cells**

Treatment with cisplatin, carboplatin and oxaliplatin, using two concentrations of 10 and 20 μm with the time period of 15 minutes, the initial response of the treated breast cancer cells started with the formation of pores on the cell membranes indicating the active process of drug influx/efflux. The pores on the surface of the MDA-MB-231 cells were deeper and wider due to the high number of lamellipodia, unlike the two cell types; MCF-7 and BT-474. Subsequently the lamellipodia retracted causing the cells to shrink and change their shape to semioval and to round shape. This was more evident to a higher extent in the MDA-MB-231 cells.

When we treated all the three types of breast cancer cells for 12 hours with the three types of PBDs, SEM revealed the early stages of apoptosis presented by convoluted membrane, membrane blebs and apoptotic bodies. The membrane blebbing is caused by deep cytoskeleton rearrangement as result of alterations in organelle distribution and cell shape, a pattern of apoptosis. Differences on the response of the cells to the three types of PBDs were detected for BT-474 and MCF-7 cells. BT-474 cells sensitivity response was maximal for Carboplatin whereas MCF-7 cells sensitivity response was maximal for cisplatin. However, MDA-MB-231 cells response was similar for all the PBDs. Hence, cell mediated drug response is dependent on the cellular characteristic and the drug action.

leading to the activation of calpain-1 [33]. Calpains belong to a family of Ca2+-dependent proteases which play many roles in basic cellular processes including cell proliferation and apoptosis, through activation of the caspase pathways. Calpain-1 and calpain-2, encoded by CAPN1 and CAPN2, respectively, are the most abundant isoforms within their family [31]. Although we, and others, have shown that cisplatin-induced apoptosis occurs by way of the calpain-1 dependent pathway, [34–36]; however, information in TNBC cells is limited. This prompted us to investigate the role of the calpain-1 pathway by way of the endoplasmic retic-

Triple-Negative Breast Cancer, Cisplatin and Calpain-1 http://dx.doi.org/10.5772/intechopen.74657 151

Using Von Koss staining, we were able to represent the variation of Ca2+ deposits between the cisplatin-treated and untreated TNBC cells. Ca2+ deposits in the cytoplasm increased with increasing cisplatin concentration (0, 20 and 40 μm) in the cisplatin-treated cells with no sig-

Several studies have concentrated on the investigation of non-nuclear pathways in the apoptosis of cancer cells induced by cisplatin [31, 32, 34]. Such studies contribute to the understanding of the causes of sensitivity and resistance to cisplatin [31, 37]. The endoplasmic reticulum is involved in the regulation of cellular responses to stress and alterations in Ca2+ homeostasis [38]. Alterations in Ca2+ homeostasis and accumulation of misfolded proteins in the endoplasmic reticulum caused endoplasmic reticulum stress resulting in apoptosis [39]. Using TEM, we detected the intracellular deposits of cisplatin and its structural changes on the endoplasmic reticulum in TNBC cells. TEM micrographs revealed that cisplatin induced clear structural changes in both the endoplasmic reticulum and the mitochondria. This phenomenon represented swelling of the lumen and disarrangement of their internal folding as compared to the control cells without treatment which appeared as well-defined structures. Hence, these findings were consistent with a study conducted by Mandic et al. who demonstrated that the endoplasmic reticulum is the non-nuclear target

Studies have reported that calpain-1 is mainly located in the cytoplasm of breast cancer cells [40, 41]. We also used immunohistochemical staining to confirm this finding. The staining intensity of calpain-1 in the cytoplasm increased with increasing concentrations (0, 20 and

The results of some experiments attempted to investigate the role of calpain-1 in the apoptotic death of TNBC cells induced by cisplatin by way of the endoplasmic reticulum are summa-

**3.4. Cisplatin activated calpain-1 and induced apoptosis through the endoplasmic** 

**3.2. Cisplatin caused structural changes in the endoplasmic reticulum of TNBC cells**

ulum in the apoptotic death of TNBC cells induced by cisplatin.

**3.1. Cisplatin caused calcium release in TNBC cells**

nificant deposits observed in the untreated cells.

of cisplatin [31].

40 μm) of cisplatin.

rized in **Table 1**.

**reticulum-mediated pathway**

**3.3. Location of calpain-1 in TNBC cells**

### **2.3. Effect of PBDs on the intracellular organelles of breast cancer cells**

We then used TEM to gain further insight into the ultrastructural alterations induced by PBDs and to study how the drug cytotoxicity differentially caused these alterations. Other distinct morphological characteristics of apoptosis consistent with the literature were evident such as shrinkage of the cytoplasm, microvilli retraction, fragmentation and condensation of the nucleus and swelling of both the mitochondria and endoplasmic reticulum [24, 25]. Splitting of apoptotic cells characterizes the final stage of apoptosis [24]. In addition to apoptosis, TEM micrographs also revealed the necrotic type of death. Changes identified on plasma membrane shows incoherence, causing cell swelling and organelles disruption. Occasionally, apoptotic cells, *in vitro*, undergo a late process of secondary necrosis. Necrosis was considered to be a physical process of cell death that was not regulated. However, emerging evidence suggests that it is as another form of apoptosis and an independent genetically encoded cell death pathway [25, 26]. Overall, treated cells with the three types of PBDs exhibited similar ultrastructural changes exhibiting distinct features such as the increased number of vacuoles portraying as a defense mechanism for cell survival and this is consistent with other studies in other types of cancers [27–29]. PBD deposits were mainly attracted to the fat droplets of the cells suggesting an active role of cellular lipids in the potentiation of PBDs to induce apoptosis.

Few but prominent differences between the three types of breast cancer cells were detected when treated with PBDs. These included the following;

