ANGPTL3 overcomes sorafenib resistance via suppression of SNAI1 and CPT1A in liver cancer

Figures

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  Fig. 1. ANGPTL3 is lower expressed in HCC tissues and correlated with good prognosis in HCC patients. (A) Venn diagram of differentially expressed genes identified in two online available datasets (GSE14520 and GSE94550). Levels of ANGPTL3, MTTP, AMBP, RELN and SLC4A3 in GSE14520 (B) and GSE94550 (C) were analyzed. Kaplan-Meier analysis of overall (D) and recurrence-free survival (E) based on the indicated gene expression in GSE14520. Median expression levels of those genes were used as the cutoff. Survival was analyzed using the log-rank test. (F) Expression levels and survival outcomes of ANGPTL3 in TCGA database were analyzed using the GEPIA tool (http://gepia.cancer-pku.cn/). **, P < 0.01.

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  Fig. 2. ANGPTL3 expressions are lower expressed in sorafenib-resistant liver cancer cell lines. (A) Cell viability was assayed in parental liver cancer cells- and sorafenib resistant cell lines (HepG2SR, Huh7SR and J7SR)-treated with/without sorafenib. In addition, the IC50 values of sorafenib for the indicated cell lines were shown. (B) ANGPTL3 expression was measured in liver cancer cell lines, as indicated, via qRT-PCR analysis. GAPDH was used as endogenous control.

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  Fig. 3. ANGPTL3 suppresses cell migration and enhanced sorafenib-induced cell death. (A) ANGPTL3 expression in human hepatocyte (HH) and liver cancer cell lines was detected by western blot analysis. GAPDH served as endogenous control. The intensity of ANGPTL3 was quantified and normalized to GAPDH, with relative fold-changes compared to those in HepG2 cells. (B) Stable overexpression of ANGPTL3 was established in Huh7, J7, and Mahlavu cell lines, while stable knockdown of ANGPTL3 was achieved in the Hep3B cell line. The expression levels of ANGPTL3 in vector control (vc) and ANGPTL3-stable cell lines were assessed by western blot analysis. The ANGPTL3 signal was quantified and normalized to GAPDH, with relative fold-changes compared to the vc group. (C) The cell migration in ANGPTL3 stable cell lines was evaluated by Transwell migration assay. The migrated cells were stained by crystal violet and quantified by Image J software. (D) Conditioned medium (CM) from ANGPTL3-overexpressing Mahlavu cell cultures was collected and used for treating liver cancer cells as indicated. The cell migration of liver cancer cells was detected by Transwell migration assay. (E) The cell viability was…

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  Fig. 4. ANGPTL3 induces lipid peroxidation of liver cancer cell lines (A) The cell motility in the indicated liver cancer cell lines was evaluated by Transwell migration assay. The migrated cells were stained by crystal violet and quantified by Image J software. (B) The cell viability was determined by AlamarBlue cell viability reagent in ANGPTL3-overexpressing HepG2SR cell lines treated with/without sorafenib. The lipid peroxidation in ANGPTL3 stable cell lines (C) and sorafenib resistant cells (D) was assessed by lipid peroxidation assay. *, P < 0.05; **, P < 0.01.

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  Fig. 5. SNAI1 expression is negatively regulated by ANGPTL3 (A-B) MRNA levels of SNAI1 in ANGPTL3 stable cell lines were determined via qRT-PCR. GAPDH was used as endogenous control. (C) Protein levels of SNAI1 in the ANGPTL3-overexpressing Mahlavu cell lines were measured via western blot analysis. GAPDH was used as endogenous control. (D) The correlation between ANGPTL3 and SNAI1 in TCGA dataset was analyzed via Pearson correlation.

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  Fig. 6. ANGPTL3 facilitates CPT1A protein degradation in sorafenib-resistant liver cancer cell lines. (A) The protein levels of CPT1A in the indicated cell lines were measured via western blot analysis. vc: vector control group. GAPDH was used as endogenous control. The fold-changes of CPT1A in the specified cell lines were quantified and presented. (B) CPT1A expression in ANGPTL3-overexpressing cell lines, as indicated, with or without MG132 treatment was detected via western blot analysis. GAPDH was used as endogenous control. The fold-changes of CPT1A in the specified cell lines were quantified and presented. (C) The turnover rate of CPT1A protein was detected in cycloheximide (CHX) chase experiments. The intensity of CPT1A was calculated and normalized to the endogenous control, GAPDH.