Quercetin combined with shTERT induces apoptosis in ovarian cancer via the P53/Bax pathway, and RGD-MSN/QR/shTERT nanoparticles enhance the therapeutic efficacy

Abstract

Background: Ovarian cancer (OC) is a highly malignant gynecological tumor with poor current treatment effects. Telomerase reverse transcriptase (TERT) is an important component of telomerase and plays an important role in the progression of ovarian cancer. Quercetin(QR) has been shown to inhibit the cell cycle and induce the apoptosis in various types of tumors. However, the mechanism of quercetin in ovarian cancer and whether it can be applied in the treatment of ovarian cancer has not been fully understood.

Results: OC cells were intervened with QR in vitro and it was found that QR only inhibited the cell cycle but not induced cell apoptosis. By conducting network pharmacology, proteomics and TCGA-OV database analysis, we found that QR inhibited the cell cycle by binding to P53 and P21. However, in this study, overexpressed TERT in OC could bind to P53 and inhibit the binding of QR to P53, failing to induce tumor cell apoptosis. After TERT was knocked down, QR significantly suppressed the cell cycle of OC cells and induced apoptosis.To realize high drug delivery efficiency and drug targeting to improve the effect of inhibiting OC, we designed and prepared RGD-MSN/QR/shTERT nanoparticles for the combined administration of QR and shTERT. As confirmed by the in vivo experiments, RGD-MSN/QR/shTERT possessed good targeting ability and significant OC inhibiting effect, with no adverse reactions, and improved the survival benefits.

Conclusions: This study demonstrated the mechanistic and therapeutic advantages of combining QR with shTERT in the treatment of OC. Based on this mechanism, we synthesized the novel nanoparticles (RGD-MSN/QR/shTERT) and verified the favorable OC inhibiting effect in vivo, providing a novel strategy for the treatment of OC.

Keywords: Nanoparticle; Ovarian cancer; P53; Quercetin; TERT.

Scheme 1.

Schematic illustration of RGD-MSN/QR/shTERT preparation and suppression of OC by activating the P53/Bax pathway

Fig. 1

Quercetin inhibits the cycle of ovarian cancer cells and unable to induce apoptosis. A The effect of different concentrations of quercetin on the proliferation level of ovarian cancer cells was measured by CCK-8; (B) The effect of different concentrations of quercetin on the proliferation level of ovarian cancer cells was tested by cell clone formation assay; (C) The effect of different concentrations of quercetin on ovarian cancer cell proliferation was tested by EDU assay; (D) The effect of different concentrations of quercetin on the ovarian cancer cell cycle was measured by flow cytometry; (E) The effect of various concentrations of quercetin on apoptosis of ovarian cancer cells was determined by flow cytometry; (F) The effect of different concentrations of quercetin on apoptosis in ovarian cancer cells was examined by the TUNEL assay. ^*p < 0.05, ^***p < 0.001

Fig. 2

Quercetin inhibits ovarian cancer via the P53 pathway by network pharmacological analysis. A Wayn diagram of targets of active components in quercetin and associated targets in ovarian cancer; B PPI networks of the intersection genes; C KEGG enrichment analysis of intersection genes; D GO enrichment analysis of intersection genes; E P53 pathway suppresses the cell cycle and induces apoptosis; F Molecular docking of quercetin to P53; G Molecular docking of quercetin to P21; H Expression level of P53 pathway protein after different concentrations of quercetin intervention by Western Blot in ovarian cancer cells. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001

Fig. 3

Quercetin unable to induce apoptosis in ovarian cancer was analyzed by proteomics and TCGA database. (A) Quercetin non-intervention/intervention of mice and tumor xenografts in the CDX model constructed by ES-2; (B) Quercetin non-intervention/intervention of mice and tumor xenografts in the CDX model constructed by SKOV-3; (C) The effect of non-intervention/intervention of quercetin on tumor volume in the CDX model constructed by ES-2; (D) The effect of non-intervention/intervention of quercetin on tumor volume in the CDX model constructed by SKOV-3; (E) Histogram of differential proteins without intervention/intervention of quercetin in the CDX model constructed by ES-2; (F) Histogram of differential proteins without intervention/intervention of quercetin in the CDX model constructed by SKOV-3; (G) Volcanic map of differential proteins without intervention/intervention of quercetin in the CDX model constructed by ES-2; (H) Volcanic map of differential proteins without intervention/intervention of quercetin in the CDX model constructed by SKOV-3; (I) Wayn diagram of upregulated differential proteins in the CDX model constructed by ES-2 and SKOV-3; (J) Wayn diagram of downregulated differential proteins in the CDX model constructed by ES-2 and SKOV-3; (K) KEGG enrichment analysis of differential proteins in the CDX model constructed by ES-2; (L) GO enrichment analysis of differential proteins in the CDX model constructed by ES-2; (M) KEGG enrichment analysis of differential proteins in the CDX model constructed by SKOV-3; (N) GO enrichment analysis of differential proteins in the CDX model constructed by SKOV-3; (O) Heatmap of the visualized coexpression of TP53 with other genes from the TCGA database; (P) TP53 and TERT expression correlation analysis from the TCGA database; (G) Protein molecular docking of P53 and TERT; (H) CO-IP experiments detect P53 and TERT binding

Fig. 4

Quercetin combine with shTERT induced apoptosis in ovarian cancer in vitro. (A) TERT knockdown of the ovarian cancer cell lines ES-2 and SKOV-3; (B) The effect of quercetin combine with shTERT on the proliferation level of ovarian cancer cells was measured by CCK-8; (C) The effect of quercetin combine with shTERT on the ovarian cancer cell cycle was measured by flow cytometry; (D) The effect of quercetin combine with shTERT on apoptosis of ovarian cancer cells was determined by flow cytometry; (E) Expression level of P53 pathway protein after quercetin combine with shTERT intervention by Western Blot in ovarian cancer cells. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001

Fig. 5

Preparation and characterization of RGD-MSN/QR/shTERT. (A) Schematic diagram of RGD-MSN/QR/shTERT synthesis; (B) Hydrodynamic size distribution of RGD-MSN, RGD-MSN/QR and RGD-MSN/QR/shTERT by DLS; (C) Zeta potential of RGD-MSN, RGD-MSN/QR and RGD-MSN/QR/shTERT; (D) TEM images of RGD-MSN, RGD-MSN/QR and RGD-MSN/QR/shTERT; (E) The release kinetics of QR and shTERT from RGD-MSN/QR and RGD-MSN/QR/shTERT at pH 7.4 and 37 ℃; (F) Stability of naked shTERT and RGD-MSN/QR/shTERT against RNase measured by agarose gel electrophoresis; (G) CLSM images of ES-2 and SKOV-3 cells incubated with PBS, Free Cy5-shTERT and RGD-MSN/QR/shTERT for 4 h. Scale bar: 50 µm; (H) The bioluminescence images of tumour-bearing mice 12 h after injection of PBS, naked Cy5-shTERT and RGD-MSN/QR/shTERT to observe their targeting ovarian cancer ability

Fig. 6

The effect of RGD-MSN/QR/shTERT against inhibition in ovarian cancer in vivo. (A) The intervention flow chart, n = 5; (B, C&D) The effect of PBS, Quercetin combine with shTERT, RGD-MSN, RGD-MSN/QR and RGD-MSN/QR/shTERT on tumor size in tumour-bearing mice; (E) The effect of PBS, Quercetin combine with shTERT, RGD-MSN, RGD-MSN/QR and RGD-MSN/QR/shTERT on the expression level of P53 pathway protein by immunohistochemical stain in tumour-bearing mice. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001