## 5 Discussion
Therefore, reducing its expression may help in reduction of these stem-like cells and result in lesser tumor heterogeneity. ITG\(\beta\)4 binds directly to laminin protein which in turn activates PI3K/AKT pathways and promotes proliferation, invasion, and survival of cells [64]. Protein expression study by Western blot technique showed total and phosphorylated AKT and MAPK expression was reduced upon \(\beta\)4 integrin siRNA transfection, in all the three cell lines (Figure 8a-c). Reduction in the expression of this integrin reportedly reduces the lung metastasis in aggressive breast cancers [64]. Reduction in protein expression without decrease in cell viability indicates that, in cells such as MCF-7 and 4T1, other pathways are actively involved in causing the upregulation of this gene. For example, ER\(\alpha\) signaling plays an essential role in upregulating ITG\(\beta\)4 signaling. Research done by [PERSON] et al. (2016) showed estrogen enhances the viability of breast cancer cells by activating ER\(\alpha\)-\(\Delta\)NP63-integrin \(\beta\)4 signaling. Additionally, cross talk with various growth factors, and activation of Ras homolog family member A (RhoA)/Rho-associated protein kinase 2 (ROCK-2) signaling cascades could enhance the cell motility and viability in vitro [64]. Even though no reduction in cell viability was observed in 4T1 cells, the tumor regression model showed significant decrease (\(p<0.05\)) in the tumor volume compared to CA control (Figure 11) and smaller tumor at the end of the treatment (Figure 12e). This indicates targeting this integrin and reduced signaling of vital cell proliferation pathways such as AKT and MAPK pathways play vital role in regulating \(\beta\)4 expression and its tumor induced modelling. While various cross talks are involved between the signaling molecules and several upstream and downstream molecules are targeted, one of the ways by which integrins cooperate with AKT and ERKis by suppressing cyclin-dependent kinase inhibitor p21/cyclin dependent kinase inhibitor p27 jointly, which causes shift in the balance of forkhead box O3 (Foxo3a), an essential transcriptional activator for apoptosis, from the nucleus to cytosol [65]. Similarly, it plays role in hampering of pathways involved with cell cycle, thereby causing a significant reduction in tumors. Integrin \(\lx@sectionsign\)5 (ITG\(\lx@sectionsign\)5), when highly expressed, participates in tumor growth and angiogenesis. After delivering ITG\(\lx@sectionsign\)5 siRNA along with CA nanoparticles and incubating the cells for 48 h, MTT assay revealed a decrease in cell viability of -63% in MCF-7 (Figure 6a) with -21% cytotoxicity (Table 2), -84% in MDA-MB-231 (Figure 6b) with slight cytotoxicity of -8% (Table 3), and cell viability of -84% in 4T1 cells (Figure 6c) with cytotoxicity of -7% (Table 4). Protein expression was reduced for both phosphorylated and total AKT and MAPK in MCF-7 and 4T1 cells (Figure 8a,c respectively), while in MDA-MB-231 no knockdown was seen (Figure 8b). Variation in the protein expressions may be due to different phenotypic and genotypic characteristics of cells, and while PIJK-ERK serves as one of the pathways which contribute to signaling via this integrin, several other pathways such as Src-FAK, MEK may be involved in regulating the cell viability in MDA-MB-231 cells [28]. At the end of the study, tumor regression showed significant (\(p<0.05\)) reduction in tumor volume compared to CA control (Figure 11), with smaller tumor in comparison with CA control (Figure 12f), indicating successful delivery of target antagonist via CA delivery vehicle. Recent studies have shown integrin \(\lx@sectionsign\)5 contributes to TGF-\(\beta\)-induced EMT and resistance to radiotherapy and chemotherapy in various stages of breast cancer [28]. Therefore, by sequestering this molecule, besides targeting AKT and MAPK oncogenic proliferation pathways, vital EMT pathways could also be affected.