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

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 summarized in **Table 1**.


stress indicator proteins such as GRP78 and calmodulin suggest the involvement of endoplasmic reticulum stress-dependent Ca2+ release in the cellular mechanism of action of cisplatin. The ability of cisplatin-induced apoptosis by way of endoplasmic reticulum stress has been shown to involve calpain-mediated activation of caspase-12 [42]. Caspase-12 is localized to the endoplasmic reticulum and may be activated by the disturbance of intracellular calcium homeostasis [43]. Cyclopiazonic acid (CPA) is a selective Ca2+ ATPase inhibitor, which depletes the endoplasmic reticulum (ER) of Ca2+ and therefore, activates Ca2+ − dependent proteases such calpain. For that reason, the activity of calpain-1 was enhanced by CPA through the endoplasmic reticulum-mediated pathway which further increased the TNBC cells response to cisplatin-induced apoptosis. In contrast, the sensitivity was attenuated by calpain-1 inhibition using the exogenous inhibitor, calpain-1 siRNA. These findings support the role of calpain-1 responsible for the pro-apoptotic effects of cisplatin in TNBC cells by way of endoplasmic reticulum. Hence, targeting calpain-1 activity with specific inhibitors could be a novel approach in limiting development of primary tumors and formation of

Triple-Negative Breast Cancer, Cisplatin and Calpain-1 http://dx.doi.org/10.5772/intechopen.74657 153

TNBC has been reported to have a clinical and pathological aggressive pattern due to its heterogeneous characteristic [44]. The ineffectiveness of hormonal and targeted therapies and poor prognosis for this subtype requires developing alternative therapeutic strategies such as biomarkers. The expression of a number of proteins has been shown to be associated with clinical outcome in TNBC patients [40, 45, 46]. Hence, there is a need to identify additional biomarkers to allow personalized treatment for patients with TNBC. For this reason, we explored the role of calpain-1 as a potential prognostic factor for TNBC therapy. We also evaluated the proliferation and apoptotic index for their potential use as possible prognostic factors since the biological behavior of tumor growth is a result of a balance between the proliferative activity and the number of cells dying by apoptosis [47]. Thus, they are considered as dominant histopathologic features in tumors. Several studies have also shown that calpain-1 expression significantly associated with tumor grade [40], proliferation [48, 49] and apoptosis [50]. Therefore, we also assessed the association between calpain-1 expression, cell

We tested calpain-1 protein expression and the proliferative/apoptotic index on paraffinembedded tissues from a cohort of 55 patients with TNBC. The main histological type was infiltrative ductal carcinoma in 96.4% (53 of 55), infiltrative lobular carcinoma in 1.8% (1 of 55) and micropapillary carcinoma 1.8% (1 of 55). Patients were females with a median age of 47 years (19–74). A total of 34 cases (61.8%) were premenopausal with no family history of breast cancer. Based on the disease indexing system, half (50.9%) of the patients were defined as stage III or IV at the time of diagnosis. Almost half of the patients ( = 26, 47.3%)

**4. Calpain-1 as a potential prognostic factor in TNBC**

proliferation and apoptosis in TNBC tissues.

**4.1. Patient characteristics**

metastases.

Apoptosis was measured by Hoechst staining using fluorescent microscopy.

**Table 1.** Summary of results of 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.

We have shown in this study the effect of cisplatin on calpain-1 protein and its activation in TNBC cells. This has also been reported by others in other types of cancer cells [34, 35]. The finding that the increase in both calcium deposits and upregulation of endoplasmic reticulum stress indicator proteins such as GRP78 and calmodulin suggest the involvement of endoplasmic reticulum stress-dependent Ca2+ release in the cellular mechanism of action of cisplatin. The ability of cisplatin-induced apoptosis by way of endoplasmic reticulum stress has been shown to involve calpain-mediated activation of caspase-12 [42]. Caspase-12 is localized to the endoplasmic reticulum and may be activated by the disturbance of intracellular calcium homeostasis [43]. Cyclopiazonic acid (CPA) is a selective Ca2+ ATPase inhibitor, which depletes the endoplasmic reticulum (ER) of Ca2+ and therefore, activates Ca2+ − dependent proteases such calpain. For that reason, the activity of calpain-1 was enhanced by CPA through the endoplasmic reticulum-mediated pathway which further increased the TNBC cells response to cisplatin-induced apoptosis. In contrast, the sensitivity was attenuated by calpain-1 inhibition using the exogenous inhibitor, calpain-1 siRNA. These findings support the role of calpain-1 responsible for the pro-apoptotic effects of cisplatin in TNBC cells by way of endoplasmic reticulum. Hence, targeting calpain-1 activity with specific inhibitors could be a novel approach in limiting development of primary tumors and formation of metastases.
