## 4 Discussion  
Chemotherapy resistance remains a challenge, despite the availability of several well-known chemotherapeutic agents [30]. Considering the growing interest in natural compounds as potential cancer treatments, we extensively researched the molecular mechanisms unpinning the anti-cancer properties of BSN in HCC cells. We found that BSN inhibited the STAT3/JAK2 signaling pathway activity in HCC cell lines by reducing cell viability and causing apoptosis, which was regulated by ROS (Figure 1). Additionally, our results aligned with previous studies showing that BSN inhibited cell proliferation and colony formation in HCC and lung cancer [5; 31]. Increased TUNEL-positive cells, increased caspase-3 cleavage, and PARP cleavage followed BSN treatment and showed pro-apoptotic properties. Bcl-2 inhibited apoptosis by acting as an anti-apoptotic regulator (Figure 6). There was much evidence that Bcl-2 suppressed apoptosis [32; 33; 34]. In our study, Bcl-2 activation was dramatically inhibited by BSN. Our findings aligned with previous research [8; 35]. The BSN treatment released cytochrome C, which triggered caspase cascades, resulting in apoptosis [5]. Western blot analysis confirmed the caspase-mediated cell death induced by BSN was inhibited by z-VAD-FMK, a pro-caspase inhibitor (Figure 1D). Figure 6: BSN induced apoptosis in hepatic carcinoma cells. (**A**,**B**), TUNEL staining for in situ apoptosis detection in HepG2 and C3A cells. Apoptotic-positive cells from three samples were averaged. Scale bar = 50 μm. (**C**,**D**) BSN was added to HepG2 and C3A cells (2 \(\times\) 10\({}^{6}\)/mL) for 48 h, and the indicated dose intervals; after that, whole-cell extracts were prepared, and 40 μm of those extracts were separated on a 10–12% SDS-PAGE, Slicing membranes according to molecular mass and probing antibodies against Bcl-2, Bcl-xL, surviving, and VEGF. Experiments were conducted in triplicate. [PERSON]’s post hoc test was used in this study to analyze the data. \(p\)\(<\) 0.05 was considered significant. Graph Pad prism was used. Data are presented as the mean \(\pm\) SD. ROS production inside the cell can affect several physiological functions [15; 36]. ROS can affect DNA, proteins, and lipids, causing oxidative stress and cell death. Several studies have shown that indole phytoalexin caused apoptosis in cancer cells by boosting ROS levels [37; 38]. NAC (N-acetyl-l-cysteine) inhibits ROS-dependent apoptosis. A synthetic precursor of cysteine and glutathione, NAC, has anti-ROS properties either directly by scavenging free radicals or indirectly by increasing glutathione levels. NAC is commonly used to confirm ROSs are involved in drug-induced apoptosis [39]. In order to maintain healthy ROS levels in cells, oxygen is converted to ROS by redox reactions during respiration. In normal cells, ROS generation is out of control, which results in DNA damage caused by free radicals and can cause cancer. ROSs are also responsible for nonalcoholic steatohepatitis (NASH) to HCC [36]. The high metabolic and proliferation rates of cancer cells produce more ROS than normal cells, but high ROS generation can also kill cancer cells. For example, Walsuronoid B inhibited cell proliferation by stopping the G2/M phase via mitochondrial depolarization and induced apoptosis through ROS/p53 [5]. In prior studies, BSN has also been shown to induce ROS, modulate PI3K/AKT/mTOR, and arrest cells during EMT [31]. The mechanisms underlying ROS-induced cell death from BSN are still unclear. Based on the current study, BSN increased ROS levels up to fourfold and caused cell death. In the presence of BSN, blocking ROS production with NAC significantly increased cell viability. The NAC co-treatment recovered anti-apoptotic protein Bcl-2 and decreased pro-apoptotic protein levels, resulting in a decrease in pro-caspase-3 activity. ROS appears to play a pivotal role in causing BSN-induced apoptosis in HCC cells. The tyrosine kinase STAT3 is phosphorylated by various intrinsic and extrinsic factors.