Norcantharidin Enhances the Antitumor Effect of 5-Fluorouracil by Inducing Apoptosis of Cervical Cancer Cells: Network Pharmacology, Molecular Docking, and Experimental Validation

Abstract

The high recurrence rate of cervical cancer is a leading cause of cancer deaths in women. 5-Fluorouracil (5-FU) is an antitumor drug used to treat many types of cancer, but its diminishing effectiveness and side effects limit its use. Norcantharidin (NCTD), a demethylated derivative of cantharidin, exhibits various biological activities. Here, we investigated whether NCTD could potentiate 5-FU to induce cervical cancer cell death. To assess the cell viability and synergistic effects of the drugs, cell counting kit-8 and colony formation assays were performed using HR-HPV-positive cervical cancer cell lines. Annexin V-FITC/PI staining and TUNEL assays were performed to confirm the induction of apoptosis. The synergistic effect of NCTD on the antitumor activity of 5-FU was analyzed using network pharmacology, molecular docking, and molecular dynamics simulations. Apoptosis-related proteins were examined using immunoblotting. The combination of NCTD and 5-FU was synergistic in cervical cancer cell lines. Network pharmacological analysis identified 10 common targets of NCTD and 5-FU for cervical cancer treatment. Molecular docking showed the strong binding affinity of both compounds with CA12, CASP9, and PTGS1. Molecular dynamics simulations showed that the complex system of both drugs with caspase-9 could be in a stable state. NCTD enhanced 5-FU-mediated cytotoxicity by activating apoptosis-related proteins. NCTD acts synergistically with 5-FU to inhibit cervical cancer cell proliferation. NCTD enhances 5-FU-induced apoptosis in cervical cancer cell lines via the caspase-dependent pathway.

Keywords: HPV-positive; bioactivity; cancer; nature product; network pharmacology; synergism.

Figure 1

5-FU and NCTD inhibit cervical cancer cell proliferation. (A) The structures of 5-FU (left) and NCTD (right). (B) Cell viability was measured by the CCK-8 assay after 5-FU treatment of SiHa and HeLa cells. (C) Cell viability was measured by the CCK-8 assay after NCTD treatment of SiHa and HeLa cells.

Figure 2

The synergistic effects of NCTD, 5-FU, or in combination on the cytotoxicity of HeLa and SiHa cells. (A,B) SiHa ((A) left) and HeLa ((B) left) cells were cultured for 72 h with NCTD (12.5 and 25 μM) or 5-FU (5 and 10 μM) alone and in combination. Cell survival was measured by the CCK-8 assay. Date are shown as the mean ± SEM (n = 3): *** p < 0.001, and **** p < 0.0001 versus 5-FU group. Dot plot summary showing the CI values of NCTD and 5-FU on SiHa ((A) right) and HeLa ((B) right). The CI values were analyzed using CompuSyn software, and the CI was interpreted as follows: synergy (<1), addition (=1), and antagonism (>1). (C) Colony formation of SiHa and HeLa cells after NCTD, 5-FU, or the combination treatment.

Figure 3

The combination of NCTD with 5-FU synergistically increased the proportion of cell apoptosis in HeLa and SiHa cells. (A) Flow cytometric analysis of Annexin V-FITC/PI-stained HeLa and SiHa cells after 72 h of NCTD, 5-FU, or the combination treatment. (B) TUNEL analysis of HeLa and SiHa cells treated with NCTD, 5-FU, or the combination treatment. FITC-stained cells are green, and Hoechst-stained nuclei are in blue (Magnification, ×20).

Figure 4

Network pharmacology analysis of NCTD- and 5-FU-induced apoptosis in HeLa and SiHa cells. (A) The resulting graph of the intersection between the potential targets of A and B and apoptosis-related genes. (B) PPI network of 19 overlapping genes. (C) Identification of core genes from the 19 selected genes. (D) Molecular docking analysis of NCTD and 5-FU with 10 core proteins. (E) Binding models of NCTD and 5-FU with caspase-9, respectively.

Figure 5

(A) Cell lysates were analyzed with caspase-3 and caspase-9 antibodies by immunoblotting analysis. (B) The quantified statistical results. GAPDH was used as an internal control for total protein loading. All experiments were performed in triplicate. Date are shown as the mean ± SEM (n = 3): * p < 0.05, ** p < 0.01, and **** p < 0.0001 versus 5-FU group.