**3. Cisplatin-induced calpain-1 activation by endoplasmic reticulum in TNBC cells**

Cisplatin has been shown to induce apoptosis in enucleated cells [31, 32]. It does this by initially acting on the endoplasmic reticulum causing an increase in cytosolic calcium (Ca2+), 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 reticulum in the apoptotic death of TNBC cells induced by cisplatin.

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

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

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

**1.** Carboplatin did not cause any swelling and disarrangement of the mitochondria on the

**2.** Carboplatin-treated cells exhibited more lamellar bodies compared to cisplatin or oxaliplatin treated cells. Lamellar bodies are specialized lipid storage or secretory organelles, which have a core composed of multilamellar structure and can be surrounded by a membrane [30]. It is possible that PBDs induce lipidosis in cancer cells and cause accumulation of lamellar bodies.

**3.** Carboplatin, cisplatin and oxaliplatin caused apoptosis in all the three types of breast cancer cell lines, however, it is possible that apoptosis independent of DNA damage could have contributed to the way some of the enucleated cells of the MDA-MB-231 cells die.

**3. Cisplatin-induced calpain-1 activation by endoplasmic reticulum** 

Cisplatin has been shown to induce apoptosis in enucleated cells [31, 32]. It does this by initially acting on the endoplasmic reticulum causing an increase in cytosolic calcium (Ca2+),

on the cellular characteristic and the drug action.

150 Breast Cancer and Surgery

when treated with PBDs. These included the following;

This will be discussed further in Section 3.

**in TNBC cells**

BT-474 and the MDA-MB-231 cells as opposed to the MCF-7 cells.

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

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 significant deposits observed in the untreated cells.

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

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 of cisplatin [31].

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

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 40 μm) of cisplatin.
