## 3 Results
### Effect of Doxorubicin-Mscari comosum Extract Treatment on the UPR Pathway  
To investigate the involvement of endoplasmic reticulum (ER) stress, we analyzed the expression levels of key ER stress markers in HepG2 cells treated with doxorubicin alone or in combination with MET70. RT-qPCR analysis revealed that doxorubicin treatment decreased the expression of the chaperone BiP and the disulfide isomerase PDIA2. However, co-treatment with MET70 did not alter BiP expression and significantly increased PDIA2 expression compared to doxorubicin alone. Doxorubicin also increased the expression of the ER stress sensor DNA damage-inducible transcript 3 (CHOP), while the combination with MET70 did not significantly modify this effect (Figure 5). Moreover, the expression levels of activating transcription factors 4 (ATF4) and 6 (ATF6), two proteins acting downstream in the unfolded protein response (UPR), were  
Figure 4: Effect of doxorubicin–MET70 combined treatment on the mRNA expression of genes encoding pro-inflammatory cytokines in HepG2 cells. Cells were treated with doxorubicin (0.5 \(\upmu\)M) and MET70 (100 \(\upmu\)g/mL), either alone or in combination, for 72 h. DMSO (0.02%) was used as the vehicle control. Relative mRNA expression levels of TNF-\(\alpha\) and IL-6 analyzed by qRT-PCR were calculated using the \(2^{-\Delta\text{CI}}\) method, with \(\beta\)-actin as the endogenous reference gene. Data are presented as mean \(\pm\) SEM from three independent experiments. Statistical significance was determined using one-way ANOVA followed by Holm-Sidak correction for multiple comparisons (\({}^{*}p<0.05;^{**}p<0.01;^{***}p<0.001\)). similarly decreased by doxorubicin alone and by the combination treatment, without evidence of a synergistic effect (Figure 6). Figure 5: Effect of doxorubicin–MET70 combined treatment on the mRNA expression of genes involved in endoplasmic reticulum stress in HepG2 cells. Cells were treated with doxorubicin (0.5 uM) and MET70 (100 ug/mL), either alone or in combination, for 72 h. DMSO (0.02%) was used as the vehicle control. Relative mRNA expression levels of BiP, CHOP and PDIA2 analyzed by qRT-PCR were calculated using the 2\({}^{-\Delta\mathrm{CI}}\) method, with \(\beta\)-actin as the endogenous reference gene. Data are expressed as mean \(\pm\) SEM from at least three independent experiments. Statistical significance was assessed using one-way ANOVA followed by Holm-Sidak correction for multiple comparisons (”_p_ \(<\) 0.05; \"\" \(p\) \(<\) 0.01; \"\" \(p\) \(<\) 0.001). Figure 6: Effect of doxorubicin–MET70 combined treatment on the protein expression of ATF4 and ATF6 in HepG2 cells. Cells were treated with doxorubicin (0.5 uM) and MET70 (100 ug/mL), either  
alone or in combination, for 72 h. DMSO (0.02%) was used as the vehicle control. Protein expression levels were assessed by Western blot analysis. Representative blot and densitometric quantification are shown, with protein levels normalized to \(\beta\)-actin and expressed relative to control (set to 100%). Data are presented as mean \(\pm\) SEM from at least three independent experiments. Statistical significance was determined using one-way ANOVA followed by Holm-Sidak correction for multiple comparisons (\({}^{***}\)_p_ < 0.001). Overall, these results suggest that the addition of MET70 to doxorubicin does not significantly exacerbate or mitigate the ER stress response induced by doxorubicin in HepG2 cells. Modulation of Endoplasmic Reticulum Stress Markers by Combined Doxorubicin and Muscari comosum Extract (MET70) Treatment  
Cancer cells often exploit autophagy as a survival mechanism under conditions of excessive stress and in response to cytotoxic agents. To investigate the impact of the combined doxorubicin and _Muscari comosum_ extract (MET70) treatment on autophagy, we evaluated the expression levels of two key autophagy-related genes, LC3 and Beclin-1.