Heparin antagonizes cisplatin resistance of A2780 ovarian cancer cells by affecting the Wnt signaling pathway

Abstract

Low molecular weight heparin (LMWH), the guideline based drug for prophylaxis and treatment of cancer-associated thrombosis, was recently shown to sensitize cisplatin resistant A2780cis human ovarian cancer cells for cisplatin cytotoxicity upon 24 h pretreatment with 50 μg × mL^-1 of the LMWH tinzaparin in vitro, equivalent to a therapeutic dosage. Thereby, LMWH induced sensitization by transcriptional reprogramming of A2780cis cells via not yet elucidated mechanisms that depend on cellular proteoglycans. Here we aim to illuminate the underlying molecular mechanisms of LMWH in sensitizing A2780cis cells for cisplatin. Using TCF/LEF luciferase promotor assay (Top/Flash) we show that resistant A2780cis cells possess a threefold higher Wnt signaling activity compared to A2780 cells. Furthermore, Wnt pathway blockade by FH535 leads to higher cisplatin sensitivity of A2780cis cells. Glypican-3 (GPC3) is upregulated in A2780cis cells in response to LMWH treatment, probably as counter-regulation to sustain the high Wnt activity against LMWH. Hence, LMWH reduces the cisplatin-induced rise in Wnt activity and TCF-4 expression in A2780cis cells, but keeps sensitive A2780 cells unaffected. Consequently, Wnt signaling pathway appears as primary target of LMWH in sensitizing A2780cis cells for cisplatin toxicity. Considering the outstanding role of LMWH in clinical oncology, this finding appears as promising therapeutic option to hamper chemoresistance.

Keywords: Wnt; cancer; chemoresistance; cisplatin; tinzaparin.

Figure 1. Characterization of GAG molecular structure of A2780 and A2780cis cells

Metabolically labeled GAG isolated from A2780 and A2780cis cells (A, C) and their cultivation medium (B, D) were analyzed. (A, B) Size exclusion chromotography analysis of the ³⁵S-labeled samples indicates higher HS fractions in A2780cis cells. (C, D) Ion exchange chromatography on analysis of the ³⁵S-labeled samples eluted by a linear NaCl gradient of 0.25 - 2 mol × L⁻¹ (indicated by the grey line) display a higher charge density of the A2780cis HS. The degraded CS saccharides are indicated. The table in (E) shows the proportion of HS and CS in cell and medium fractions. Data are average of two independent experiments. (F) Expression of Sulf-2 in A2780 and A2780cis cells without pretreatment, or after a 24 h tinzaparin preincubation before cisplatin addition. Expression was analyzed 72 h after cisplatin treatment by immunoblotting. Data are shown as blot for one representative experiment (F) and as relative expression by pixel density measurements related to untreated A2780 cells (G). Data from three independent experiments.

Figure 2. Analysis of cellular syndecan expression pattern and the impact of ECM on resistance

Syndecan-1 (left panel) and -4 (right panel) in tinzaparin treated A2780 (A, B) and A2780cis (C, D) cells. The expression was analyzed by flow cytometry using specific antibodies indicating no impact of tinzaparin on syndecans. Experiments were performed in triplicates. Determination of cytotoxicity 72 h after addition of cisplatin displayed as IC₅₀ in A2780 (circles) and A2780cis (squares) cells by MTT assays. The IC₅₀ for cisplatin (E) and in combination with collagen coated wells (F) are indicated for a representative experiment. MTT assays were performed in triplicates.

Figure 3

(A) Expression of heparanase in A2780 and A2780cis cells preincubated 24 h with tinzaparin before cisplatin addition, with cisplatin or both. Expression of heparanase was analyzed 72 h after cisplatin treatment by immunoblotting using a specific antibody and shown as blot for one representative experiment and as relative expression by pixel density measurements related to untreated A2780 cells, summarized in (B) from three independent experiments. (C) Determination of cytotoxicity 72 h after addition of cisplatin displayed as IC₅₀ in A2780 (circles) and A2780cis (squares) cells by MTT assays. The IC₅₀ for cisplatin (left) and in combination with a preincubation of 5 (middle) and 10 μmol × L⁻¹ (right) roneparstat 5 h before addition of cisplatin are indicated in the figure for a representative experiment. MTT assays were performed in triplicates.

Figure 4. Analysis of Wnt-associated genes in untreated (stripes) and 24 h tinzaparin treated (squares) A2780cis cells

Fold change of transcription was normalized to untreated A2780 cells.

Figure 5

Analysis of the HSPGs glypican-3 (A) and glypican-4 (B) in A2780cis cells with a 24 h tinzaparin preincubation (50 μg × mL⁻¹). The expression was analyzed by flow cytometry using specific antibodies. Experiments were performed in triplicates. (C) Expression of glypican-3 in A2780 and A2780cis cells with a 24 h tinzaparin preincubation before cisplatin addition. Expression was analyzed 72 h after cisplatin treatment by immunoblotting using a specific antibody and shown as blot for one representative experiment and as relative expression by pixel density measurements related to untreated A2780 cells (D). Data from two independent experiments.

Figure 6. Cytotoxicity of the Wnt pathway inhibitor FH535 alone and in combination with cisplatin

Data are displayed as IC₅₀ in A2780 (circles) and A2780cis (squares) cells by MTT assays. The IC₅₀ for FH535 (A, B), cisplatin (C) and cisplatin in combination with a preincubation with 0.5 μmol × L⁻¹ FH535 1.5 h before addition of cisplatin (D) are indicated in the figure for a representative experiment. Cells were seeded in a count of 20,000 (A) and 40,000 (B, C, D) 24 h before treatment with FH535 and cisplatin. MTT assays were performed in triplicates.

Figure 7. Wnt activity of A2780 and A2780cis cells

Cells were either untreated or preincubated with tinzaparin 24 h before cisplatin addition. Luciferase activity was analyzed 72 h after treatment by luminescence measurement and normalized to untreated A2780 cells. Data from three independent experiments.

Figure 8

(A) Expression of TCF4 in A2780 and A2780cis cells with a 24 h tinzaparin preincubation before cisplatin addition. Expression was analyzed 72 h after cisplatin treatment by immunoblotting using a specific antibody and shown as blot for one representative experiment and as relative expression by pixel density measurements related to untreated A2780 cells (B). Data from three independent experiments.