3.1 Curcumin treatment and ATG3 knockdown significantly inhibit cell migration in both SiHa and HeLa cervical cancer cell lines.
The results demonstrate the inhibitory effects of curcumin and ATG3 knockdown (ShATG3) on the migration of cervical cancer cells (SiHa and HeLa) using wound healing assays. Figure 2A and C show representative images of wound closure at 0, 24, and 48 hours for SiHa and HeLa cells, respectively. Both curcumin treatment and ATG3 knockdown impaired cell migration, with the combination treatment showing the most pronounced effect. Figure 2B and D provide quantitative analyses of wound closure in SiHa and HeLa cells at 24 and 48 hours. Compared to the control group, both ShATG3 and curcumin treatments significantly reduced wound closure, with the combination of ShATG3 and curcumin showing the most pronounced effect. Highlighting the potential of curcumin and ATG3 inhibition as therapeutic strategies in cervical cancer by reducing cell migration.
3.2 Curcumin treatment and ATG3 knockdown significantly increase LC3 expression in both SiHa and HeLa cervical cancer cell lines.
Explore the effects of curcumin and ATG3 knockdown (ShATG3) on LC3 expression in cervical cancer cells (SiHa and HeLa) using immunofluorescence staining and western blot analysis. Figure 3A shows representative immunofluorescence images of LC3 in SiHa cells, with DAPI staining the nuclei (blue) and LC3 (green) marking the autophagosomes. The merged images indicate the localization of LC3 within the cells. Figure 3B presents the quantification of the relative fluorescence intensity of LC3 and the relative protein levels of LC3 in SiHa cells. The results show a significant increase in LC3 fluorescence intensity in the curcumin (****P < 0.0001) and ShATG3+curcumin (***P < 0.001) groups compared to the control and ShATG3 (*P < 0.05) groups. Western blot analysis shows corresponding increases in LC3-II protein levels, confirming enhanced autophagy.
Figure 3C shows representative immunofluorescence images of LC3 in HeLa cells under similar treatment conditions. Figure 3D provides the quantification of the relative fluorescence intensity of LC3 and the relative protein levels of LC3 in HeLa cells. The results show a significant increase in LC3 fluorescence intensity in the curcumin (****P < 0.0001) and ShATG3+curcumin (****P < 0.0001) groups compared to the control and ShATG3 (**P < 0.01) groups. Western blot analysis confirms these observations with increased LC3-II levels. These results demonstrate that curcumin enhances autophagy in cervical cancer cells, and this effect is potentiated by ATG3 knockdown. The significant increase in LC3 expression, both in fluorescence intensity and protein levels, suggests a potential therapeutic role for curcumin in modulating autophagy in cervical cancer.
3.2 Curcumin and ATG3 Knockdown Modulate Key Proteins Involved in Autophagy and Metastasis in Cervical Cancer Cells
Figure 4A shows representative western blot images and quantification of MMP2, P62, and ATG3 protein levels in SiHa cells. MMP2 protein levels are significantly reduced by ShATG3 (**P < 0.01) and curcumin (****P < 0.0001) treatments compared to control. The combination treatment of ShATG3 and curcumin shows an additive effect (****P < 0.0001). P62 protein levels are also significantly reduced by ShATG3 (**P < 0.01), curcumin (**P < 0.01), and the combination treatment (****P < 0.0001) compared to control. ATG3 protein levels are significantly reduced in ShATG3 (***P < 0.001) and ShATG3+curcumin (****P < 0.0001) groups compared to control. While Figure 4B shows MMP2, P62, and ATG3 protein levels in HeLa cells. MMP2 protein levels are significantly reduced by ShATG3 (*P < 0.05), curcumin (**P < 0.01), and the combination treatment (****P < 0.0001) compared to control. P62 protein levels show no significant changes across the groups. ATG3 protein levels are significantly reduced in ShATG3 (**P < 0.01) and ShATG3+curcumin (*P < 0.05) groups compared to control. These results highlight the potential of curcumin and ATG3 inhibition as therapeutic strategies in cervical cancer by modulating key proteins involved in autophagy and metastasis.
3.3 Curcumin shows potential binding affinity with several key proteins involved in autophagy and metastasis
Figure 5A displays 2D interaction diagrams, highlighting the hydrogen bonds and hydrophobic interactions between curcumin and each target protein. In MMP2, curcumin forms hydrogen bonds with Ala84 and other residues. In MMP9, it interacts with Glu427 among other contacts. TGF-β binding involves Lys232 and several hydrophobic contacts. Curcumin binds to autophagy-related proteins including ATG3 at Lys151, LC3 at Asp106, and P62 at Tyr128. The 3D poses (Figure 5B) illustrate how curcumin fits within the active sites and the nature of the molecular interactions. These molecular docking results suggest that curcumin can interact with multiple targets involved in autophagy and metastasis, potentially modulating their activities, and contributing to its anticancer properties.