ANGPTL3 diminishes the resistance of ovarian cancer to paclitaxel by blocking the PI3K-AKT-mTOR signaling pathway

Abstract

Angiopoietin-like protein 3 (ANGPTL3) is key in ovarian cancer (OC) cell growth and metastasis, notably by enhancing natural killer cells' capacity for inducing cell toxicity and apoptosis. However, its role in influencing chemotherapy resistance in OC remains ambiguous. In this study, we discovered a correlation between reduced ANGPTL3 levels and a less favorable outcome in OC patients using the Kaplan-Meier Plotter database. Lower levels of ANGPTL3 were detected in paclitaxel (PTX)-resistant OC tissues and cell lines via western blotting and immunohistochemistry. To investigate ANGPTL3's effects, we established SKOV3/PTX and 2780/PTX as PTX-resistant OC cell lines by incrementally increasing PTX exposure and then transfecting them with overexpress ANGPTL3 (OE-ANGPTL3) lentivirus. We conducted various assays such as CCK-8, colony formation, Edu staining, flow cytometry, and transwell to investigate the impact of ANGPTL3 on PTX resistance. Additionally, this effect was examined in a mouse subcutaneous xenograft model. Both in vitro and in vivo experiments demonstrated that ANGPTL3 overexpression mitigated PTX resistance in OC cells by inactivating the PI3K-AKT-mTOR pathway. In summary, our research reveals that ANGPTL3 enhances PTX sensitivity in OC by downregulating the PI3K-AKT-mTOR pathway. The study of this study suggest that ANGPTL3 could serve as a valuable therapeutic target for OC, signifying its clinical relevance in OC management.

Keywords: ANGPTL3; Ovarian cancer; PI3K-AKT-mTOR signaling; Paclitaxel resistance; Tumorigenesis.

Fig. 1

Reduced ANGPTL3 expression was associated with poor prognosis and chemoresistance in OC. (A) Kaplan-Meier survival analysis illustrated the OS of patients with ovarian cancer between high and low ANGPTL3 expression. (B) Kaplan-Meier survival analysis illustrated the PFS of patients with ovarian cancer between high and low ANGPTL3 expression. OS, overall survival; PFS, progression free survival; HR, hazard ratio. (C) The mRNA levels of ANGPTL3 were determined in PTX-resistant and PTX-sensitive tissues using quantitative real time PCR. (D) Representative images of ANGPTL3 protein expression via Western blot analysis in PTX-resistant (R1/R2/R3) and PTX-sensitive (S1/S2/S3) tissues (n = 3; **p < 0.01). (E) Representative immunohistochemical images are presented about ANGPTL3 expression in PTX-resistant and PTX-sensitive tissues; R, resistance; S, sensitive.

Fig. 2

ANGPTL3 was lowly expressed in PTX-resistant OC cells. (A–B) CCK-8 assay was employed to detect cell viability after PTX treatment (0, 1, 5, 10, 50, and 100 nM) in SKOV3 and 2780 cells and PTX-resistant SKOV3 and 2780 cells. (C–D) Quantitative real time PCR and (E–F) Western blot analysis were performed to determine ANGPTL3 expression in IOSE-80 cells, SKOV3 and 2780 cells as well as PTX-resistant SKOV3 and 2780 cells. Each value represents the mean ± SD from three independent experiments. **p < 0.01, ***p < 0.001, compared with IOSE8; ##p < 0.01, compared with SKOV3 or 2780.

Fig. 3

Overexpression of ANGPTL3 suppressed the proliferation of PTX-resistant OC cells. (A) Western blot analysis of ANGPTL3 in SKOV3/PTX and 2780/PTX cells after transfection with OE-ANGPTL3 or Vector plasmids. (B) Cell viability was determined by CCK-8 assay in transfected SKOV3/PTX and 2780/PTX cells. (C) Colony formation assay and (D) Edu staining were conducted to assess cell proliferation ability of transfected SKOV3/PTX and 2780/PTX cells. Each value represents the mean ± SD from three independent experiments. **p < 0.01, ***p < 0.001, compared with Vector.

Fig. 4

Overexpression of ANGPTL3 induced G0/G1 arrest and apoptosis in PTX-resistant OC cells. SKOV3/PTX and 2780/PTX cells were transfected with OE-ANGPTL3 or Vector for 48 h. (A–B) The effects of OE-ANGPTL3 transfection on cell cycle distribution were determined in SKOV3/PTX and 2780/PTX cells. The representative results of cell cycle were shown in left panel and corresponding quantification of cell cycle distribution was depicted in right panel. (C–D) The effects of OE-ANGPTL3 transfection on apoptotic rate were determined in SKOV3/PTX and 2780/PTX cells. (E) The protein expression of CDK4, Cyclin D1 and caspse-3 was detected using Western blot analysis in transfected SKOV3/PTX and 2780/PTX cells. Each value represents the mean ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, compared with Vector.

Fig. 5

Overexpression of ANGPTL3 inhibited the migration and invasion capacities of PTX-resistant OC cells. SKOV3/PTX and 2780/PTX cells were transfected with OE-ANGPTL3 or Vector for 48 h. Transwell assay was utilized to analyze cell migration (A) and invasion (B) in transfected SKOV3/PTX and 2780/PTX cells. (C) The protein levels of E-cadherin, N-cadherin and Vimentin were detected in transfected SKOV3/PTX and 2780/PTX cells. Each value represents the mean ± SD from three independent experiments. **p < 0.01, ***p < 0.001, compared with Vector.

Fig. 6

Overexpression of ANGPTL3 downregulated the PI3K-AKT-mTOR signaling pathway in PTX-resistant OC cells. The protein levels of PI3K, p-PI3K, AKT, p-AKT, mTOR, and p-mTOR were determined in SKOV3/PTX and 2780/PTX cells after transfection with OE-ANGPTL3 or Vector. Each value represents the mean ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, compared with Vector.

Fig. 7

ANGPTL3 overexpression repressed PTX resistance in OC cell in vivo. BALB/c nude mice were injected with SKOV3/PTX cells (1 × 10⁷) transfected with OE-ANGPTL3 or Vector (n = 3 per group). After one weeks, the mice were intraperitoneally administered with PTX (5 mg/kg) twice a week for 4 weeks. (A) Tumor volume was monitored continuously for 35 days. On Day 35, the xenografts were resected (B) and weighed (C). (D) The mRNA expression of ANGPTL3 was determined by quantitative real time PCR in xenograft tumor tissues. (E) The protein levels of ANGPTL3, p-PI3K, PI3K, p-AKT, AKT, p-mTOR and mTOR were determined in xenograft tumor tissues. Each value represents the mean ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, compared with Vector; TIR, tumor inhibition rate.

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