*2.7. Effect of Curcumin-Loaded PGS-NPs in Inducing Apoptosis of HeLa Cells*

The induction of apoptosis in HeLa cells treated with 0.005 and 0.01 mg/mL of PGS5.0C0.5 for 24, 48 and 72 h was then investigated. PGS5.0C0.5 provoked a dose-dependent up-regulation of genes involved in apoptosis (p53 and Bax, Figure 8a,b) and cell cycle arrest (p21, Figure 8c) compared to non-treated cells. No up-regulation was observed when HeLa cells were treated with PGS5.0Blank (data not shown). Western blot analysis confirmed that PGS5.0C0.5 increased cleaved caspase-3 and PARP levels and induced apoptosis (Figure 8e). A significant reduction of the transcript levels of the viral HPV E6 oncogene was also observed, suggesting an inhibitory effect of PGS5.0C0.5 on its expression (Figure 8d). Overall, these findings suggest that PGS5.0C0.5 NPs are capable of inducing apoptosis by activating cell cycle arrest and apoptosis in HeLa cervical cancer cells.

**Figure 8.** Effect of curcumin-loaded PGS-NPs on the induction of apoptosis. Real-time RT-PCR analysis of transcript levels of p53 (**a**), p21 (**b**), Bax (**c**) and HPV E6 genes (**d**) in HeLa cells treated with 0.005 or 0.01 mg/mL of PGS5.0C0.5 for 24, 48 and 72 h. Each transcript level is expressed as fold change (fc) with respect to control samples treated with vehicle (CNT). Values are means ± SEM (n = 6) (**e**) Western blot panel and (**f**) relative quantification of cleaved caspase-3 and PARP levels in HeLa cells treated with 0.005 or 0.01 mg/mL of PGS5.0C0.5 for 72 h. Data were analyzed with ImageJ and normalized to α-tubulin and expressed as fold change over CNT. Values are means ± SEM (n = 3). \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001 indicate significant differences (twoway ANOVA followed by Tukey's multiple comparisons test) versus CNT. CNT (blank): Control; PGS5.0C0.5 (light orange) [PGS] 0.005 mg/mL [Curcumin] 0.0005 mg/mL; PGS5.0C0.5 (dark orange) [PGS] 0.01 mg/mL [Curcumin] 0.001 mg/mL).
