Baicalein improves the chemoresistance of ovarian cancer through regulation of CirSLC7A6

Abstract

Purpose: The present study aimed to investigate whether baicalein improves the sensitivity of resistant ovarian cancer cells to cisplatin.

Methods: Transcriptomic sequencing and bioinformatics analysis were used to screen differentially expressed CirSLC7A6 in A2780 and A2780/CDDP cells. RT-qPCR was performed to examine the expression levels of CirSLC7A6, miR-2682-5p, and SLC7A6. Cell proliferation and apoptosis were examined using a Cell Counting Kit-8 assay and flow cytometry, and cell migration and invasion were analyzed using wound healing and Transwell assays. Cell suspensions were inoculated into the subcutaneous tissues of the bilateral interscapular region of nude mice. Saline, cisplatin, baicalein and cisplatin plus baicalein were intraperitoneally injected to observe the effects on tumor growth. Toxicity analyses in the liver and kidney were performed using H&E staining. RT-qPCR and immunohistochemistry were used to detect the expression of CirSLC7A6, miR-2682-5p, and SLC7A6 in tumor tissues, and western blot analysis was carried out to measure protein expression levels.

Results: CirSLC7A6 was markedly upregulated in A2780/CDDP cells compared with the A2780 cells. CirSLC7A6 knockdown notably increased the expression of miR-2682-5p and decreased SLC7A6 expression. The rates of inhibition and apoptosis in the group treated with a combination of cisplatin and baicalein were significantly higher than those of the cisplatin and baicalein groups of A2780/CDDP shCirSLC7A6 cells. In A2780/CDDP shCirSLC7A6 cells, migration and invasion were significantly higher in the cisplatin and baicalein groups, compared with the combined treatment group. In the A2780/CDDP shCirSLC7A6 cell xenograft, the tumor weight of the combined treatment group was significantly lower than that of the cisplatin and baicalein groups. In addition, the combination of cisplatin and baicalein did not induce higher levels of toxicity in the liver or kidney. Baicalein alone and in combination with cisplatin notably reduced the expression of CirSLC7A6 and SLC7A6, and increased the expression of miR-2682-5p in the A2780/CDDP shCirSLC7A6 cell xenograft. In A2780/CDDP shCirSLC7A6 cells, the expression levels of P-Akt, P-mTOR, P-Erk, Bcl-2 and MMP2 were lower in the combined treatment group than in the control group.

Conclusions: Treatment with baicalein improved the sensitivity of ovarian cancer cells to cisplatin and inhibited cell proliferation, metastasis and tumor growth.

Keywords: Baicalein; Chemoresistance; CirSLC7A6; Ovarian cancer; SLC7A6.

Fig. 1

CirSLC7A6 may affect the chemoresistance of ovarian cancer through regulating miR-2682-5p and downstream gene SLC7A6. A Network of circRNAs and genes in A2780 and A2780/CDDP cells. B GO and KEGG pathway analysis. C Heatmap. D Binding sites of CirSLC7A6 and miR-2682-5p. Reverse transcription-quantitative PCR was carried out to detect the expression of CirSLC7A6, miR-2682-5p and SLC7A6 in (E) A2780 and A2780/CDDP cells, (F) A2780/CDDP shGFP and A2780/CDDP shCirSLC7A6 cells. And (G) A2780 Vector and A2780 CirSLC7A6 cells. circRNA, circular RNA; miRNA, microRNA; SLC7, solute carrier family 7; GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes

Fig. 2

CirSLC7A6 knockdown increases the anti-proliferative and pro-apoptotic effects of the combined treatment of cisplatin and baicalein on resistant ovarian cancer cells. A Chemical structure of baicalein. B Baicalein inhibits the proliferation of T29 cells. C Cisplatin inhibits the proliferation of six ovarian cancer cell types. D Baicalein inhibits the proliferation of six ovarian cancer cell types. E Comparison of the inhibition of six ovarian cancer cell types treated with the same drug. F Comparison of the inhibition of six ovarian cancer cell types treated with different drugs. G Comparison of the apoptosis rates of six ovarian cancer cell types treated with the same drug. H Comparison of the apoptosis rate of A2780 cells treated with different drugs. I Comparison of the apoptosis rate of A2780/CDDP cells treated with different drugs. J Comparison of the apoptosis rate of A2780/CDDP shGFP cells treated with different drugs. K Comparison of the apoptosis rate of A2780/CDDP shCirSLC7A6 cells treated with different drugs. L Comparison of the apoptosis rate of A2780 Vector cells treated with different drugs. M Comparison of the apoptosis rate of A2780 CirSLC7A6 cells treated with different drugs. circRNA, circular RNA; SLC7, solute carrier family 7; shRNA, short hairpin RNA

Fig. 3

Baicalein suppresses the migration and invasion of resistant ovarian cancer cells through regulating CirSLC7A6. A Intercellular comparison of the migration rate. B Intracellular comparison in A2780 and A2780/CDDP cells. C Intracellular comparison in A2780/CDDP shGFP and A2780/CDDP shCirSLC7A6 cells. D Intracellular comparison in A2780 Vector and A2780 CirSLC7A6 cells. E Intercellular comparison of cell invasion. F Intracellular comparison in A2780 and A2780/CDDP cells. G Intracellular comparison in A2780/CDDP shGFP and A2780/CDDP shCirSLC7A6 cells. H Intracellular comparison in A2780 Vector and A2780 CirSLC7A6 cells. circRNA, circular RNA; SLC7, solute carrier family 7; shRNA, short hairpin RNA

Fig. 4

CirSLC7A6 knockdown enhances the synergistic inhibitory effects of cisplatin and baicalein on the tumor growth of ovarian cancer. A Tumors subcutaneously transplanted with A2780 cells. B Tumors subcutaneously transplanted with A2780/CDDP cells. C Tumors subcutaneously transplanted with A2780/CDDP shGFP cells. D Tumors subcutaneously transplanted with A2780/CDDP shCirSLC7A6 cells. E–F Cisplatin plus baicalein causes no injury in the liver or kidney of mice transplanted with A2780/CDDP shCirSLC7A6 cells. circRNA, circular RNA; SLC7, solute carrier family 7; shRNA, short hairpin RNA

Fig. 5

Reverse transcription-quantitative PCR indicated that the expression levels of CirSLC7A6 were significantly decreased, and a combination of cisplatin and baicalein downregulated the expression of CirSLC7A6 in the A2780/CDDP shCirSLC7A6 cell xenograft. A Intercellular comparison. B Intracellular comparison. C-D Immunohistochemical results demonstrated that the expression levels of SLC7A6 were significantly decreased and the combined treatment of cisplatin and baicalein downregulated the expression of SLC7A6 in the A2780/CDDP shCirSLC7A6 cell xenograft. circRNA, circular RNA; SLC7, solute carrier family 7; shRNA, short hairpin RNA

Fig. 6

Baicalein inhibits the proliferation of resistant ovarian cancer cells through regulating CirSLC7A6. A Protein bands. B The quantitative histograms of the intercellular comparison between A2780 and A2780/CDDP cells. C The quantitative histograms of the intercellular comparison between A2780/CDDP shGFP and A2780/CDDP shCirSLC7A6 cells. D The quantitative histograms of the intracellular comparison in A2780 and A2780/CDDP cells. E The quantitative histograms of the intracellular comparison in A2780/CDDP shGFP and A2780/CDDP shCirSLC7A6 cells. Baicalein inhibits the metastasis of resistant ovarian cancer cells through regulating CirSLC7A6. F Protein bands. G The quantitative histograms of the intercellular comparison. H The quantitative histograms of the intracellular comparison. circRNA, circular RNA; SLC7, solute carrier family 7; shRNA, short hairpin RNA