## 4 Discussion
Moreover, a higher energy level of the external electric field acting on the biomembrane makes it easier to surpass the electroporation energy barrier, leading to the electroporation and expansion of pore radius through the influence of the electric field force [44; 45]. The cellular force-electric response to pulsed electric fields (PEFs) has been extensively studied as a crucial approach to investigate the different cell death pathways under electric field pulse stress. For instance, [PERSON] et al. [46] applied esPEF with a pulse width of 11 ns to cells and observed weakened microtubule polymerization, along with changes in zeta potential and microtubule structure. Similarly, [PERSON] et al. [47] treated tobacco BY-2 cells with esPEF of 10 ns pulse width, resulting in the depolymerization of cortical actin, the detachment of fibrous actin bundles from the cell periphery, and their contraction towards the nucleus. Cell migration and invasion are triggered by various chemotactic agents, which, upon binding to cell surface receptors, activate intracellular signaling pathways regulating actin cytoskeleton reorganization. Invasive protrusions or filopodia-like structures are generated by cancer cells to invade the extracellular matrix and migrate to distant sites, necessitating significant actin microfilament polymerization [48]. The depolymerization and geometric changes of cytoskeletal proteins under PEF were also confirmed in this study. Under nsPEF treatment, the anchoring between the actin layer and the plasma membrane was disrupted, resulting in their separation. Concurrently, the actin layer depolymerization and fibrous actin disruption increased the cell elasticity and aggravated the strain on various cellular components when subjected to force [49; 50], akin to the effects of actin polymerization inhibitors [30]. In the experiment presented in this article, the fluorescence intensity of the cell nucleus significantly decreased following nsPEF treatment, indicating the successful penetration of the high-frequency pulsed electric field into the cell and its impact on the nucleus. This resulted in damage to the nuclear membrane and the disruption of the nuclear matrix. Researchers at the Center for Bioelectrics at Old Dominion University in the United States have employed pulsed electric fields with pulse widths ranging from 10 to 300 ns to treat cells and observed that the nuclear membrane becomes charged and forms electroporation pores. Furthermore, the pulsed electric field alters the structure of the nuclear protein complex, induces double-strand breaks in DNA, disrupts cell transcription mechanisms, and promotes cell apoptosis [51; 52; 53]. However, literature reports also suggest that nuclear membrane electroporation facilitates the entry of DNA plasmids into the nucleus, thereby enhancing the gene expression in cells [30]. Hence, it becomes essential to control nsPEF pulse parameters to strike a balance between cell killing by pulsed electric fields and gene transfection, which remains a significant scientific question for the further advancement of nsPEFs. Mitochondria are vital organelles that play a crucial role in providing cellular energy through aerobic respiration. They are also involved in significant processes such as cell differentiation, signal transduction, and apoptosis regulation. The findings of this study demonstrate that treating MDA-MB-231 cells with nsPEFs resulted in the dissipation of mitochondrial membrane potential, the disruption of the double membrane structure, and release of cytochrome c from the mitochondria. This release of cytochrome c activated the apoptotic protein caspase, leading to apoptosis [27; 54]. Additionally, the permeabilization of the mitochondrial membrane resulted in ATP depletion, which acts as a critical damage-associated molecular pattern (DAMP) and triggers immunogenic cell death, including necrosis [55; 56].