## 4 Discussion  
Hepatocellular carcinoma (HCC), the most common form of liver cancer, accounts for approximately 75-85% of all liver cancer cases. Despite advancements in treatment strategies, HCC remains a major clinical challenge, with a five-year survival rate of only 8.37% [27, 28]. A promising strategy for improving cancer therapy involves the use of phytochemicals as adjuvants to conventional chemotherapy, due to their demonstrated anti-tumor, anti-inflammatory, and anti-angiogenic properties [16, 17, 18, 19, 21, 22]. In this study, we evaluated for the first time the potential of a methanol-water extract of _Muscari comosum_ (MET70) to enhance the efficacy of doxorubicin in HepG2 liver cancer cells. The combination treatment significantly reduced cell viability compared to either agent alone, indicating a synergistic cytotoxic effect and the possibility of using lower doses of doxorubicin to mitigate its systemic toxicity. Although limited studies have evaluated _M. comosum_ in cancer models [24, 25], previous reports have highlighted its high polyphenol and flavonoid content, which are known to be associated with anticancer effects [17, 19, 21, 22]. Doxorubicin exerts its cytotoxic effects through multiple mechanisms, including the induction of reactive oxygen species (ROS) [7, 8, 9, 10, 11, 29]. Interestingly, MET70 alone did not significantly alter ROS levels in our model, but when combined with doxorubicin, a substantial increase in ROS production was observed. This synergistic effect may occur due to the downregulation of antioxidant defenses, including glutathione peroxidase-1 (GPX-1) and superoxide dismutase (SOD2), as well as the potential interference with the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway, as well as reduced levels of mitochondrial uncoupling protein 2 (UCP2). This enhanced ROS production likely contributed to the increased cytotoxicity observed following the combination treatment [30, 31, 32, 33]. Although MET70 alone upregulated Nrf2 and did not significantly increase ROS, the combination treatment with doxorubicin suppressed antioxidant defenses (e.g., GPX-1, SOD2), potentially explaining the observed ROS synergy and reduced viability. While cancer cells can tolerate moderate ROS through adaptive mechanisms, excessive ROS surpasses antioxidant capacity, resulting in oxidative damage and cell death. Thus, MET70 may potentiate doxorubicin efficacy by disrupting redox homeostasis beyond a tolerable threshold. It is well established that elevated levels of pro-inflammatory cytokines can further promote ROS generation, creating a feedback loop that exacerbates oxidative stress [33]. Oxidative stress and inflammation are closely linked with ROS promoting the release of pro-inflammatory cytokines. Doxorubicin activates NF-kB and increases IL-6 and TNF-\(\alpha\) levels [10, 34, 35]. We found that co-treatment with MET70 increased IL-6 expression, suggesting that MET70 may modulate inflammatory responses during chemotherapy. Similar behavior has been noted for polyphenol-rich extracts that both exacerbate or attenuate cytokine responses, depending on the context [18]. Although IL-6 can promote tumorigenesis in chronic inflammation, its acute elevation in response to high ROS levels may facilitate cancer cell death. We also investigated markers of the unfolded protein response (UPR), which is activated during ER stress, particularly under conditions of proteotoxicity induced by chemotherapy. As expected, doxorubicin modulated several ER stress markers and reduced the expression of the chaperone BiP and protein disulfide isomerase PDIA2 while increasing the expression of the pro-apoptotic ER stress sensor CHOP. Additionally, doxorubicin decreased the expression of the downstream UPR mediators ATF4 and ATF6. Given the cytotoxic synergy observed between MET70 and doxorubicin, it was initially hypothesized that MET70 would exacerbate ER stress responses.