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.

Glycosaminoglycans (GAGs) are linear polysaccharides comprised of glucosamine/uronic acid-containing repeating disaccharides with different sulphation and uronic acid epimer patterns. 8 GAGs are covalently linked to proteins during their biosynthesis.
The proteins that carry GAG chains are known as proteoglycans. to cell but also from tissues to tissues. 4,10 The biological functions of proteoglycans are largely dependent on their GAG chains. 7,11 Rapid turnover 11,12 and structure diversity generated by both biosynthesis and post biosynthesis modification allow GAGs to promote or inhibit a myriad of signalling pathways, 11 such as fibroblast growth factor (FGF) and FGF receptor (FGFR) signalling pathway. [13][14][15][16][17] Indeed, it has been well documented that GAGs play critical roles in physiological and pathological processes in both human and animals. 4,[18][19][20][21][22][23][24][25][26][27] Properly sulphated GAGs are indispensable for their biological activities. Thus, it is highly desirable to have small molecules that disrupt GAG biosynthesis in cell culture conditions or in animal models to establish GAG structure/function relationship. At present, only sodium chlorate, an inhibitor of the universal sulphate donor 3-phospho-adenyl-5-phosphosulfate (PAPS), 28,29 and D-xylosides, which act as artificial primers of GAG biosynthesis, [29][30][31] has such function.
Therefore, it is important to develop proper assays to screen for reagents that disrupt GAG biosynthesis.
All BLMs are positively charged molecules and cannot get inside of cells by free diffusion. 4 GAGs are present at the cell surface in the form of proteoglycans. Interestingly, proteoglycans are well-established cell surface endocytosis receptors. 32,33 Therefore, we hypothesized that positively charged BLMA5 may interact with the negatively charged GAGs of proteoglycans on the cell surface for their cellular uptake. Indeed, by treating six different cell lines including a unique Chinese hamster ovary cell mutant defective in cell surface GAG biosynthesis (CHO745) with BLM A2, B2, A5 or combination of any two of them, 1 the cytotoxicity data suggested that GAGs might be involved in the cellular uptake of all BLMs. 1,4 Since BLM has no optical properties, we have synthesized a series of small molecules with intrinsic fluorescence with or without positive charge, that is isothiouronium-or bromo-modified curcumin-pyrimidine analogs. 4 We found that only the positively charged curcumin-pyrimidine analogs have Golgi localization once inside of the cells. 34 As such, we assumed that BLMA5 might interfere with GAG biosynthesis in Golgi.

Changes in CS disaccharide compositions of proteoglycans in
BLM-treated rat model have been reported. 35 By using a novel stable isotope labelling coupled with LC/MS method, we found BLM inhibits both HS and CS sulphation in cancer cell lines. 4 In current study, human colon cancer cell lines HCT116 and HT29, human lung cancer cell lines H1299 and A549, a Lewis lung carcinoma-injected C57BL/6 mouse model were used to test if BLMA5 had the same effect. Wild-type Chinese hamster ovary cell line CHOK1, cell surface   GAG-deficient CHO cell line CHO 745 36 and 3-O-sulphotransferase-1-expressing CHOK1 cell line, CHO 3.1, 37 were also used to test how   the cell surface GAGs were related to the cytotoxicity effects of   BLMA5. Furthermore, by using flow cytometry-based cell surface FGF/FGFR/GAG binding assay plus stable isotope labelling coupled with LC/MS analysis, we demonstrated that BLMA5 inhibited glycosaminoglycan sulphation in the tumour tissues of the mouse model.

| Cell growth inhibition assay
The method was similar to the previously published one. 4

| The effects of different types of GAGs on the cytotoxicity induced by BLMA5 in cell culture
The method was similar to the previously published one. 4

| The effects of sodium chlorate in cell culture media on the cytotoxicity induced by BLMA5
The method was similar to the previously published one. 4 Briefly, sulphation was inhibited by sodium chlorate treatment. 28

| Purification of GAGs from control and BLMA5 treated cells
The method is the same as described previously. 4 Briefly, control cells (0.1% DMSO treated) and BLMA5 treated cells with a final DMSO concentration of 0.1% were used for GAG preparations.
GAGs were then extracted from the control and BLMA5 treated cells.

| Establishing C57BL/6 mouse model of lung tumours
All mice were treated in accordance with the regulations approved by the Animal Ethics Committee of Ocean University of China, which were performed in accordance with National Institutes of Health guidelines.
The method is similar to that published in previous study. 38 LLC cells (Type culture collection of the Chinese academy of sciences) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum plus ampicillin and streptomycin routinely, and incubated in 5% CO 2 at 37°C. The LLC cells (1 × 10 7 cells/mL) were then injected into C57BL/6 mice to prepare enough tumour cells to establish C57BL/6 mouse model of lung tumours. Briefly, the tumour tissues from Lewis lung carcinoma mice were triturated and the tumour cell suspensions were then prepared in that the cell concentration was adjusted to 1 × 10 7 cells/mL, and 0.2 mL cell suspensions were injected subcutaneously into the armpit of right anterior superior limbs of female C57BL/6 mice (National Institute for the Control of Biological and Pharmaceutical Products) to establish the lung tumour model, the mice were 6-8 week old (weight 18.6 ± 0.5 g). Three days later, the mice were randomized into two groups (16 mice per group), a treatment group injected with BLMA5 in saline (1.2 mg/kg) into the hind leg muscle every 3rd day.
A control group injected with the same volume of saline every 3rd day. All treatments lasted for 28 days. Mice were killed, and lung tumours were harvested and stored at −80°C until used.

| Purification of GAGs from lung tumours
Tumours were defatted before GAG purification. For short, tumours were weighed and homogenized in cold acetone and shook at shaking table for 24 hours. The acetone was removed by aspiration after centrifugation at 3939 g for 15 minutes. Methanol and chloroform were then added to pellets while stirring to a final ratio of 1:2 (CH 3 OH:CHCl 3 v/v) and left overnight at room temperature followed by centrifugation at the same condition to obtain the defatted tumour tissues, which were washed twice with ethanol and dried at room temperature. The defatted tissues were suspended in 10 mL 0.25 mol/L NaCl buffer (0.25 mol/L NaCl, pH 6.0, 20 mmol/L NaAc, 0.01% Triton X-100) before protease digestion using the identical GAG purification procedure described above.

| Generating HS and CS disaccharide by enzymatic digestion
The method is the same as described previously. 4 Briefly, GAGs were divided into two parts, one for HS disaccharide analysis and the other for CS disaccharide analysis. For HS digestions, GAGs were incubated with 0.33 mU each of heparin lyase I, II and III. For CS digestions, GAGs were incubated in digestion solution containing chondroitinase ABC. Digestion was monitored by using a Spectra MAX M2 plate reading spectrophotometer.

| BLMA5 treatment changed FGF/FGFR/GAG binding on the cell surfaces of A549 and HCT116 cells detected by flow cytometry
We have previously developed a flow cytometry-based cell surface binding assay that allows to measure the interaction between cell surface GAGs with fluorescence-labelling GAG binding proteins,

F I G U R E 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)

| Cell surface GAGs were responsible for getting BLMA5 inside cells to exert its cytotoxicity
Sodium chlorate is the reversible sulphation inhibitor, 44

| Changes of CS and HS disaccharides in A549 and HCT116 cells
The data in Figures 2 and 3 indicated BLMA5 treatment changed GAG structures of A549 and HCT116 cells. However, detailed GAG structure characterization is required to demonstrate such changes.
In GAG research field, CS and HS disaccharide compositional analysis is used for such purpose. To facilitate quantitative comparison of GAG disaccharide compositions from different cell types, we have previously tagged the reducing end of enzyme-digested disaccharides with aniline-containing normal and stable isotopes. 4,45 Because different isotope tags have no effect on LC retention times but are discriminated by MS analysis, differentially isotope-tagged disaccharides can be compared simultaneously by LC/MS. 4,45,46 To test how BLMA5 changed biosynthesis and metabolism of  TA B L E 2 Molecular masses for GAG disaccharides detected in cancer cells exactly observed in the MS data ( Figure 4). We then summarized all the molecular mass over charge (m/z) for the stable isotope labelled GAGs disaccharides of BLMA5-treated or non-BLMA5-treated cancer cells in Table 2.

| The CS and HS disaccharide compositions from cancer cells
In this study, each disaccharide was separated by using a solvent gradient (See details in Section 2). The ion current intensities of the stable isotope labelled disaccharides were increased with the increase of acetonitrile concentrations during the LC run. Therefore, we used commercial disaccharides to obtain the response factor for each disaccharide using the same method we reported previously. 4,11 Then, the proportion of each GAGs disaccharide was calculated based on MS data. The results were shown in Figure

| BLMA5 treatment reduced both HS and CS sulphation in lung tumours of LLC-injected C57BL/7 mice
The LC/MS analysis revealed that BLMA5 not only suppressed HS sulphation but also changed the quantity and sulphation pattern of CS in both A549 and HCT116 cancer cells. The next question we asked was if BLMA5 also changed HS and CS biosynthesis in lung tumours in a well-established C57BL/6 mouse model. 38 To this end, C57BL/6 mouse model of lung tumours was established. 38 Three days later, the mice were divided randomly into two groups (16 mice per group), each mouse in the treatment group was injected with BLMA5 dissolved in saline and each mouse in the control group was injected with the same volume of saline (See details in Section 2). All mice were killed at 28th day, and lung tumours were harvested and weighed. As expected, BLMA5 treatment inhibited tumour growth and resulted in fewer tumours with smaller size (0.22 ± 0.16 g) compared to that of saline-injected control mice (0.66 ± 0.46 g). Most importantly, the sulphated disaccharides in both HS and CS were decreased in the BLMA5-treated group compared to that of salinetreated control group. The CS disaccharide compositions of BLMA5treated and saline-treated mice were shown in Figure 6A, and the HS disaccharide compositions of BLMA5-treated and saline-treated mice were shown in Figure 6B, respectively. The fold quantity differences in each CS ( Figure 6C) and HS ( Figure 6D) disaccharides from lung tumours between BLM-treated and saline-treated mice were calculated based on the data presented in Figure 6A,B, that is the PMP-labelled disaccharide% from lung tumours of BLMA5-treated C57BL/6 mice was divided by the D5PMP-labelled disaccharide% from lung tumours of saline-treated C57BL/6 mice. The CS D0a6 and D0a10 disaccharides ( Figure 6C) were significantly decreased in lung tumours of BLMA5-treated mice. Overall sulphation of HS disaccharides ( Figure 6D) was also decreased in BLMA5-treated mice. Thus, the data in Figure 6A-D revealed that BLMA5 treatment reduced both CS and HS sulphation in the lung tumours of C57BL/6 mouse model significantly.

| D ISCUSS I ON
We In Figure 5B, BLMA5 treatment led to a dramatic increase in CS D0a6 in HCT116 cells; however, D0a6 in BLMA5 treated LLC cells ( Figure 6A) was significantly reduced, which raised the question how could BLMA5 have opposite effects in these two cell lines.  cell-based assays. 41,48 Among all the GAGs, heparin is the mostly charged and also has the rare 3-O-sulphated sequences that are critical for its anticoagulant activities. Heparin is the most active GAG in most of biological tests but with exceptions. 49 Indeed, the data in Figure 4 showed that heparin appeared less effective than HS in inhibiting BLMA5-induced cytotoxicity, indicating HS was involved in the uptake of BLMA5.

| Statistical analysis of data
All statistical calculations were done using IBM SPSS Statistics 21.
All data are represented as the mean ± SD t test was used to determine the possible significant differences (P < .05) of indicators between control group and treatment groups.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

AUTH O R CO NTR I B UTI O N S
YL and LZ designed the study. YL, XL, YL and YH performed the experiments and analysed the data. CH, HW, JL, GZ, SZ and AZ contributed reagents/materials/analysis/interpretation of the data. YL and LZ wrote the paper.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data in the current study are available from the corresponding authors on reasonable request.