Pingyangmycin inhibits glycosaminoglycan sulphation in both cancer cells and tumour tissues

Abstract

Pingyangmycin is a clinically used anticancer drug and induces lung fibrosis in certain cancer patients. We previously reported that the negatively charged cell surface glycosaminoglycans are involved in the cellular uptake of the positively charged pingyangmycin. However, it is unknown if pingyangmycin affects glycosaminoglycan structures. Seven cell lines and a Lewis lung carcinoma-injected C57BL/6 mouse model were used to understand the cytotoxicity of pingyangmycin and its effect on glycosaminoglycan biosynthesis. Stable isotope labelling coupled with LC/MS method was used to quantify glycosaminoglycan disaccharide compositions from pingyangmycin-treated and untreated cell and tumour samples. Pingyangmycin reduced both chondroitin sulphate and heparan sulphate sulphation in cancer cells and in tumours. The effect was persistent at different pingyangmycin concentrations and at different exposure times. Moreover, the cytotoxicity of pingyangmycin was decreased in the presence of soluble glycosaminoglycans, in the glycosaminoglycan-deficient cell line CHO745, and in the presence of chlorate. A flow cytometry-based cell surface FGF/FGFR/glycosaminoglycan binding assay also showed that pingyangmycin changed cell surface glycosaminoglycan structures. Changes in the structures of glycosaminoglycans may be related to fibrosis induced by pingyangmycin in certain cancer patients.

Keywords: cancer; glycosaminoglycan; heparan sulphate; mass spectrometry; pingyangmycin.

Figure 1

Growth inhibitory effect of BLMA5 on three CHO cell lines and four different cancer cell lines. Two human lung cancer cell lines A549 and H1299, two human colon cancer cell lines HCT116 and HT29, and three Chinese hamster ovary cell lines (CHO745, CHOK1 and CHO3.1) were used to measure the percentage of viable cells after 48 h exposure to 0‐160 μmol/L BLMA5. The experiment was repeated three times with similar results. The untreated cells (control) were assigned values of 100, and the results were presented as mean ± SD (n = 3). Significance: *P < .05 vs 0 μmol/L group

Figure 2

FGF/FGFR/GAG ternary complex formation on the cell surface of BLMA5 treated (10 μmol/L, 2 h) cancer cells. A, A549 cells, B, HCT116 cells

Figure 3

Cell surface GAGs were responsible for getting BLMA5 inside cells to exert its cytotoxicity. (a) Sulphation inhibition by sodium chlorate reduced cytotoxicity effects of BLMA5 on cancer cell lines. Human lung cancer cell line A549 and human colon cancer cell line HCT116, and Chinese hamster ovary cell line CHO745 were used to measure the percentage of viable cells after exposure to varying concentrations of sodium chlorate (0.016‐10 mmol/L) with or without BLMA5 (15 μmol/L). The experiment was repeated three times with similar results. The untreated cells (control) were assigned values of 100, and the results were presented as mean ± SD (n = 3). (b) Soluble GAG reduced cytotoxicity of BLMA5 in both HCT116 and A549 cells. Human lung cancer cell line A549 and human colon cancer cell line HCT116, and Chinese hamster ovary cell line CHO745 were used to measure the percentage of viable cells after 48 h exposure to varying concentrations of soluble GAG (0.15, 1.5, 15 μmol/L) in the presence of BLM (15 μmol/L) or BLM treatment alone (15 μmol/L). The GAGs used in the experiment include CS‐A, CS‐B, CS‐C, CS‐D, CS‐E, HS and heparin as described earlier.44 Since different GAGs have different molecular weight distributions and BLMA5 is a small molecule comparable to the size of a repeating disaccharide unit in the GAGs, the disaccharide concentration for each GAG was calculated based on the knowledge of its average mass of the repeating disaccharide unit. The experiment was repeated three times with similar results. The untreated cells (control) were assigned values of 100, and the results were presented as mean ± SD (n = 3). Significance: *P < .05 vs BLMA5 group

Figure 4

MS data of six GAG disaccharides from the PMP and D5PMP‐labelled samples from A549 cells. HS and CS disaccharides from enzymatically digested GAGs isolated from BLMA5‐treated or control A549 cells were tagged with PMP (blue, BLMA5‐treated, 80 μmol/L 4 h) or D5PMP (red, the control, 0 μmol/L 4 h), respectively. The two independently labelled samples were proportionally admixed to ensure that each sample was from the same amount of cell‐proteins. The co‐injected mixture was then subjected to LC/MS analysis. Since each disaccharide can be tagged with two molecules of PMP or D5PMP, each co‐eluted pair of PMP‐ and D5PMP‐labelled disaccharide should have a molecular weight difference of 10 in theory, which was exactly observed in the MS data shown above. All disaccharides were further identified by directly comparing both LC elution positions and m/z data with that of PMP‐labelled, commercially available disaccharide standards (See details in Section 2)

Figure 5

Disaccharide compositions of HS and CS from BLM A5‐treated and untreated A549 and HCT116 cells. Disaccharides from enzymatically digested HS and CS isolated from BLM A5‐treated and untreated HCT116 and A549 cells were tagged with PMP or D5PMP, respectively. The eight sets of PMP‐ or D5PMP‐labelled samples were proportionally admixed to ensure equal cell‐protein loading into four samples: A, CS disaccharide compositions of A549 cells with or without BLM A5 treatment; B, CS disaccharide compositions of HCT116 cells with or without BLM A5 treatment; C, HS disaccharide compositions of A549 cells with or without BLM A5 treatment; D, HS disaccharide compositions of HCT116 cells with or without BLM A5 treatment. The disaccharide compositions were calculated based on the MS data (mean ± SD from three independent measurements, Significance: *P < .05 vs control group). BLM A5 treatment conditions: HCST (80 μmol/L 4 h) and LCLT (10 μmol/L 48 h)

Figure 6

BLM A5 treatment reduced both HS and CS sulphation in lung tumours of LLC‐injected C57BL/7 mice. Disaccharides from enzymatically digested HS and CS isolated from lung tumours of BLM A5‐treated or saline‐treated LLC‐injected C57BL/6 mice were tagged with PMP or D5PMP, respectively. Samples were proportionally admixed to ensure they were from equal weight of lung tumours (BLM A5 treated groups: n = 16; control group: n = 16). A, CS disaccharide compositions of lung tumours from saline‐treated or BLM A5‐treated C57BL/6 mice; B, HS disaccharide compositions of tumours from saline‐treated or BLM A5‐treated C57BL/6 mice. C, The changes in CS disaccharides in saline‐treated vs BLM A5‐treated C57BL/6 mice; D, The changes in HS disaccharides in saline‐treated vs BLM A5‐treated C57BL/6 mice. Data are expressed as mean ± SD from three independent measurements. *P < .05 vs control group, statistical significance was determined by t test

Figure 7

Summary of the major discoveries. D0a0, D0a6, D0a4, D0A0, D2A0, D0H6, D0S0, D2H0, D0S6 and D2S0 represent ΔUA‐GalNAc, ΔUA‐GalNAc6S, ΔUA‐GalNAc4S, ΔUA‐GlcNAc, ΔUA2S‐GlcNAc, ΔUA‐GlcN6S, ΔUA‐GlcNS, ΔUA2S‐GlcN, ΔUA‐GlcNS6S and ΔUA2S‐GlcNS, respectively