Evaluating Tumor-Associated Activity of Extracellular Sulfatase by

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Evaluating Tumor-Associated Activity of Extracellular Sulfatase by Analyzing Naturally-Occurring Substrate in Tumor Microenvironment of Hepatocellular Carcinoma Yue Yu, Hao Li, Yucai Yang, Yitao Ding, Zhaoxia Wang, and Genxi Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03469 • Publication Date (Web): 22 Nov 2016 Downloaded from http://pubs.acs.org on November 24, 2016

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Analytical Chemistry

Evaluating Tumor-Associated Activity of Extracellular Sulfatase by Analyzing Naturally-Occurring Substrate in Tumor Microenvironment of Hepatocellular Carcinoma Yue Yu†, Hao Li‡, Yucai Yang§, Yitao Ding†,*, Zhaoxia Wang§,*, Genxi Li‡, #, * † ‡

. Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing 210008, China.

. State Key Laboratory of Pharmaceutical Biotechnology and Collaborative Innovation Center of Chemistry for Life Sciences, Department of Biochemistry, Nanjing University, Nanjing 210093, China.

§

#

. Department of Oncology, The Second Affiliated Hospital of Nanjing Medical University, Nanjing 210011, China.

. Center for Molecular Recognition and Biosensing, School of Life Sciences, Shanghai University, Shanghai 200444, China.

ABSTRACT: The progress of cancer is intimately connected with the activity of the extracellular (ECM) enzymes. To evaluate the promoting effect of these enzymes on tumor development in pathological bio-context, we propose in this work to analyze their natural substrates in the ECM. This strategy is demonstrated by studying heparan sulfate (HS), the substrate of ECM sulfatase, in the development of hepatocellular carcinoma (HCC). An assay is designed to study the abundance and sulfation of HS, and to evaluate the interactions between HS and the growth factors, such as fibroblast growth factor 2 (FGF2). Peptides derived from the amyloid peptide and various growth factors are employed to detect HS and evaluate their affinity towards the growth factors, while Ruthenium polypyridyl complex is taken as a photocatalyst to achieve more sensitive signal readout. Applying this method to HepG2 cells, correlated changes between the activity of sulfatase 2 in regulating FGF2 induced cell proliferation, and the abundance, degree of sulfation and growth factor binding of HS can be observed. This method has also been applied to analyze clinical tissue samples of HCC. The results may suggest tumor progress-related alterations in the above studied biochemical features of HS. These results may point to the prospect of using this method to facilitate the diagnosis and prognosis of HCC in the future.

The development of cancer is an extremely complicated pathological process. This process involves intricate cross-talk between the tumor cells and their extracellular matrix (ECM) 1, 2 . These ECM-cell interactions are strongly influenced by the activity of ECM enzymes3-6. For instance, ECM sulfatase controls the sulfation of heparan sulfate (HS), an ECM polysaccharide that can bind with growth factors 7, so as to control their bioavailibity to the resident tumor cells 8. Activity of this enzyme may modify the side chains of HS, resulting in lowered affinity towards growth factors 8. The subsequent release of growth factors may start uncontrolled proliferation, leading to neoplastic transformation. In fact, suppression/promotion of cancer via the activity of sulfatase is a recognized molecular mechanism in the development of various cancers9-14, such as prostate, pancreatic, breast cancer and hepatocellular carcinoma (HCC). From the above described example, it can be seen that activity of ECM enzyme is not restricted to simple degradation of ECM components, but can involve elaborate side chains modifications that can reconfigure the ECM-cell interactions to promote tumor. Therefore, if those aspects in connection with the tumor promoting context can be evaluated by the activity of ECM enzyme, the timely evaluation of the onset and progression of cancer may be greatly facilitated. For this task, existing methods may be restricted by the adoption of artificial substrate probes15-18. First, the application

of artificial probe in biological and clinical samples will inevitably result in blending of the artificial and the natural substrates, so that the result represents only part of the activity of the target enzyme. Second and more importantly, the artificial probe commonly contains only the substrate sequence, but without the other motifs that may also be influenced by the activity of the enzyme, as in the case of the natural substrate. So, the important effects of enzyme activity, in connection with the pathological context, as described above, cannot be assayed with artificial probes. To achieve more bio-context relevant evaluation of ECM enzyme activity in the development of cancer, we propose direct bioanalysis of natural substrate from the sample. And this is demonstrated in studying the sulfation and interactions of HS in the development of HCC. Specifically, a bioassay is developed for the detection of HS, the degree of its sulfation, as well as its affinity towards the growth factors. The bioassay is designed taking advantage of the interactions of HS with various species. First, the various growth factors, ECM proteins and even amyloid peptides19, can specifically recognize HS7, 20. Moreover, through miniaturization and sequence abstraction, these ligands have generated shorter peptide sequences that can be chemically synthesized and modified19, 21-23. This enables us to employ the abstracted peptide as probes to detect HS. And the

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Scheme 1. Principle of design. The routes a~c, a~f and a~i are separately the methods for detecting HS, assaying the degree of HS sulfation and for studying the affinity of HS towards growth factor derived peptides. Not drawn to scale. affinity towards these peptides can also to some extent represents the affinity towards the original ligands. On the other hand, to detect the degree of sulfation, the electrostatic interaction between cation such as polypyridyl Ru(II), and the polyanionic HS can be employed24. Heavily sulfated HS can attract great amount of cationic biosensing agents. For the signal generation in detecting HS, as well as in analyzing sulfation and affinity, the cathodic stripping of HS is employed25. The direct stripping of HS can give out electrochemical signal readout in proportion to the amount of HS. For studying the degree of sulfation, polypyridyl Ru(II) is employed as a photochemical catalyst26 to induce radical depolymerization of HS27, 28. The stripping of the product of depolymerization produces signal readout much larger than that of the direct stripping of HS, and this amplified signal readout is in proportion to the amount of photochemical catalyst, the amount of which is in turn, higher for more heavily sulfated HS. The various growth factorderived peptides can also interact with HS, in competence with the cation. Using this method, the affinity of differentially sulfated HS for the growth factors can be studied. The proposed method has been applied in the analysis of HS in cellular as well as in clinical samples, the detected amount of HS, its sulfation and affinity towards the growth factors, can reflect changes in biological and pathological conditions, HCC cell lines and HCC patients. These results may show the potential of the proposed method in assisting the diagnosis and administration of HCC in the future. Experimental Section Chemicals and biological materials. Peptide probe (DAEFRHDSGYQVHHQKLVFFAEDVGSNK19-

dibenzylcyclooctyne (DBCO), and growth factor derived peptides, FGF1 peptide: KKHAEKNWFVGLKKNGSCKRGPR21, FGF2 peptide: SNNYNTYRSRKYSSWYVALKR22, VEFG peptide: KTKRKRKKQRVK23 were custom-synthesized as lyophilized powder, purity>95%, by Shanghai Science Peptide Co, Ltd. Recombinant perlecan, glypican and syndecan were from R & D system. Versican and human sulf2 were from Shrbio, Nanjing. 4-azidoaniline hydrochloride was from Seebio, Shanghai. 9-mercapto-1-nonanol (MN) was bought from Sigma-Aldrich. Purified HS, molecular weight approximately, 9 kDa was from Galen laboratory supplies. [Ru(bpy)2dppz]Cl2 was synthesized following the established method29. All the other chemicals were of analytical-grade. The solutions of the peptide probe were prepared by dissolving the powder to 10 µM with 10 mM phosphate buffer solution (PBS) (pH 7.4). The solution of the various growth factor derived peptides was prepared by dissolving the power with 50 mM TBS (pH 7.5) to the desired concentrations. The standard sample of the various recombinant proteins was prepared by dissolving the lyophilized powder with 50 mM TBS (pH 7.5) to the desired concentrations, and the standard sample of purified HS was prepared by dissolving the lyophilized powder with 50 mM TBS (pH 7.5) to the desired concentrations. The solution of Sulf2 was prepared by dissolving the lyophilized powder with 50 mM TBS (pH 7.5), containing 150 mM NaCl and 10 mM MgCl2, to the desired concentrations. All solutions were prepared with double-distilled water, which was purified with a Milli-Q purification system (Branstead, USA) to a specific resistance of 18 MΩ·cm. HepG2 cell (Type Collection of Chinese Academy of Science, Shanghai) was cultured in Dulbecco modified Eagle medium, 10% fetal bovine serum, and was maintained in a humidified atmosphere

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with 5% CO2 at 37 °C. For the detection, HepG2 cells of which sulf2 was knocked down with sulf2 siRNA, oriGene, following the manufacturer’s instruction, were plated in 24 well culture plates (Nanjing Protein in Biotechnology Co.), incubated with 10 ng/mL FGF2 for 3 days. The cells were then collected, counted and diluted serially. These samples were then centrifuged at 1000 rpm for 1 min, the supernatant was used for detection. For MTT assays, the cells were seeded on 96 well plates at 3000 cells per well and incubated with 10 ng/mL FGF2 for 48 hours. Cell viability was then assessed by MTT-reducing capacity. For the detection of clinical samples of hepatocellular carcinoma, fresh tissues samples were collected from 10 patients at Nanjing Drum Tower Hospital, within 30 min of surgical resection, and after elected consent by the local ethical committee, 3 samples were also obtained from healthy donors of liver transplantations. The samples were from the primary lesion and the adjacent normal tissue. The retrieved samples were immediately sliced on ice to 1 mm3, followed by digestion with type II collagenase at 37 °C for 30 min. The resulted supernatant was centrifugated at 800 rpm for 5 min, the supernatant containing no cellular components were then retained for detection. Electrode Treatment and Modification. The glassy carbon (GC) electrode was successively polished with 1, 0.3 and 0.05µM alumina slurry on a lapping cloth, followed by being sonicated in tandem with ethanol and water, each for 5 min and dried in a stream of high purity nitrogen. Meanwhile, 4azidoaniline diazonium cation was prepared by adding 1 mg NaNO2 into 5 ml of 0.5 M HCl, 3 mM 4-azidoaniline hydrochloride. This reaction mixture was kept for 1 h in an ice bath and in darkness. The GCE was then immerged in the resulted solution and electrochemically modified (scan rate: 0.1 V/s, scanning range: from 0 V to 0.8 V vs. Ag/AgCl, 5min). After thorough rinsing and being dried with nitrogen stream, the Azide modified electrode was incubated with 10 µM DBCO linked peptide probes, in 10 mM PBS, pH 7.4 at 4 °C over night. After rinsing, the electrode was subsequently dipped in 100 µL MN solution (1 mM MN in 10 mM PBS, pH 7.4) for 3 h at room temperature. Finally, after thorough rinsing, the electrode was dried under mild nitrogen stream. Detection. The peptide-modified electrode was incubated with standard sample of the recombinant protein, the purified HS, the ECM fraction of cellular samples or fractioned clinical sample for proper time at 37 °C. Then the electrode was thoroughly rinsed with double-distilled water, followed by being dipped in 5% Tween-20 for 30 min to exclude non-specific adsorption. Following route a~c in Scheme 1, cathodic stripping is performed in 0.006 M NaCl (pH 6.8), preconcentration potential -0.02 V, duration of preconcentration, 30 s. After a period of 5 s rest, DPVs were recorded from -0.02 to 1 V. Following route a~f, the electrode was incubated with the complex cation in dark for proper time, followed by the photocleavage under UV for 5 min irradiation at 365 nM. The vial with the electrode immerged in it is then used as the electrochemical cell for cathodic stripping. Following route a~i, the complex cation and the growth factor derived factors were mixed to the concentrations as indicated on Figure 3, then the electrode was treated as above, following route a~f. Experimental Measurements. Isothermal titration calorimetry (ITC) measurements were conducted using a MicroCal

ITC200 System (GE healthcare life sciences). The titration was conducted at 25 °C. The titration schedule consisted of 38 consecutive injections of 1 µL with at least a 120 s interval between injections. Heats of dilution, measured by titrating beyond saturation, were subtracted from each data set. All solutions were degassed prior to titration. The data were analyzed using Origin 7.0 software. Electrochemical measurements were carried out on a CHI660D Potentiostat (CH Instruments) with a conventional three-electrode system: the electrode immobilized with peptide as the working electrode, a saturated calomel electrode (SCE) as the reference electrode and a platinum wire as the counter electrode. Differential pulse voltammograms (DPVs) were recorded in 10 mM PBS, pH 7.4, which was deoxygenated by purging with nitrogen and maintained under this inert atmosphere during the electrochemical measurements. Experimental parameters were as follows. Step potential: 5 mV, pulse potential: 50 mV, time step potential: 20 ms. Electrochemical impedance spectra (EIS) were recorded in 5mM [Fe(CN)6]3−/4−with 1 M KNO3. The experimental parameters were as follow. Bias potential, 0.224 V vs. SCE, amplitude, 5 mV, frequency range, 0.1 Hz ~ 10 kHz. The electrochemical data were obtained from at least three times of repetition of independent experiment. Results and Discussion Scheme 1 illustrates the principle of studying the sulfation and interactions of HS, as a method to evaluate the tumor promoting/inhibiting effect of sulfatase. Usually, HS exists in the form of a heparan sulfate proteoglycan (HSPG)30: two or three strands of HS, in the form of a helix, are attached to a core protein. In the ECM, perlecan is a major form of HSPG controlling the bioavailibity of various growth factors31. To study the sulfation of perlecan, an assay is designed. First, amyloid peptide 1~28 is immobilized on the electrode surface to capture perlecan (step a), as this peptide has high affinity towards perlecan19 (association constant in the range of 10-11 M). After capturing of perlecan from the ECM fraction of the cellular sample (step b), the amount of HS can be assayed by cathodic stripping (step c). Alternatively, the electrode surface can be further incubated with the complex cation, [Ru(bpy)2DPPZ]2+ (bpy = 2,2’-bipyridine, DPPZ = dipyrido[3,2-a:2’,3’-c]phenazine) (step d). Then cathodic stripping carried out after photo-catalyzed degradation of HS can be employed to measure the degree of sulfation (step d~f). Further, the growth factors derived peptides can be employed to compete with the complex cation in binding with the HS on the surface captured perlecan (step g). The decreased signal of cathodic stripping can then be used for evaluating the affinity of the differentially sulfated HS towards the growth factors (step g~i). The detection of perlecan and its HS content is studied first. Since purified perlecan that still retains the original conformation is not readily available, the standard samples were recombinant core protein, purified HS as well as fractioned ECM samples of HepG2 cell line. The interactions of the amyloid peptide probe with the two components of perlecan, the core protein and HS, have first been studied with isothermal titration calorimetry (ITC). From Figure S1, it can be seen that the probe peptide binds strongly with the core protein, but roughly a thousand times weaker with HS, so it may be postu-

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lated that the detection of perlecan is mainly relied on the binding with the core protein. Then, the surface immobilized peptide probe is allowed to interact with the core protein, HS and the ECM fraction of cellular samples, at gradually higher concentrations (Figure 1a~c, respectively). The electrochemical impedance in Figure 1a and the current peaks of cathodic stripping in Figure 1b, c grow in proportion with the sample concentration. For the core protein, the signal response approaches saturation around 100 pM (Figure 1a), while HS of 100 nM is needed for saturation of signal response (Figure 2b, inset). For the serially diluted cellular samples (ECM fraction), it begins to yield saturated signal readout when diluted to around 3.5×105cell/mL (Figure 1c, inset). Also in Figure 1b, c, the saturated current peaks are of very similar height. These results, together with the above ITC studied binding strength, may suggest that the HS content in the saturated cellular sample is some thousand times lower than that in the purified HS sample, since the peptide probe binds to HS around a thousand times weaker than with the core protein. Meanwhile, the core protein, from its much higher affinity towards the probe, may contribute dominantly in capturing perlecan from the cellular sample. Therefore, the detection range of perlecan in the cellular sample can be roughly calculated as from the subnanomolar range to lower than 10 pM. Meanwhile, the specificity of the assay has also been examined by studying the binding of the peptide probe with various control species (Figure 1d). It can be seen that the core protein of similar HSPGs, such as glypican and syndecan, located on the cell surface, have no such strong interaction with the probe. This is also the case of the other ECM HSPG, such as versican, so the specific detection of perlecan can be confirmed. Besides, the time required for detecting the cellular sample has also been optimized, and an incubation time of around 60 min is adopted for all the following experiments (Figure S2). The degree of sulfation is then studied using polypyridyl Ru(II) to label HS and induce its radical depolymerization. Polypyridyl Ru(II) such as [Ru(bpy)2DPPZ]2+ can specifically bind with polyanion such as DNA26 and heparin24, an analogue of HS. The radical photo-cleavage of DNA has been realized with Polypyridyl Ru(II)26. So here the recognition and photo radical depolymerization of HS by [Ru(bpy)2DPPZ]2+ is attempted. The peptide probe modified electrode, after proper incubation with the cellular sample (ECM fraction), is then allowed to interact with different concentrations of the complex cation, [Ru(bpy)2DPPZ]2+. The electrochemical response of the complex cation bound on the electrode surface, as shown in Figure 2a, grows in proportion to its concentration, and the plateau of signal response is approached at 10 µM. These results are comparable with the previous reports of this complex cation with similar affinity towards DNA26. The binding of peptide probe with the ruthenium complex does not influence the secondary structures formed (Figure S3a). UV irradiation and cathodic stripping are then applied for the electrode treated with different amount of the complex cation, and a proportional increase of response with that of cation concentration, can be observed (Figure 2b). It is also observed that the signal response is much larger than that in detecting intact HS (Figure 1b, c). This may be due to the much more free diffusion of depolymerized HS fragments. These products can accumulate on the electrode surface, free from the hindrance of amyloid peptide probe, which strongly binds to perlecan,

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thereby hindering the intact HS strands from freely approaching to the electrode surface. At higher concentration, the complex cation may cleave HS into small fragments that can diffuse more freely to the electrode surface to give larger response. Interestingly, the responses at different concentrations of the complex cation can become uniformly negligible, if the depolymerized fragments are removed by rinsing and change of buffer solution (Figure S4). So, this negligible residual response can be assigned to the few HS fragments still attaching to the core protein. This may also be applicable for the following experiments with sulfatase and growth factor peptides of various concentrations. The proper time for the incubation of the complex cation has also been optimized (Figure S5). The electrochemical impedance spectra (EIS) have also been recorded in the above steps of detecting perlecan and assaying sulfation with the complex cation. As shown in Figure 2c, the bare electrode appears as a straight line (curve a), indicating no evident impedance. Peptide modification increases the surface impedance, resulting in a half circle of moderate diameter on the spectrum (curve b). Capture of perlecan from the cellular sample drastically increases the impedance (curve c), the morphology of the surface at this step has also been captured using AFM (Figure S3b). A control is also conducted using the recombinant core protein sample of 100 pM, which concentration has been calibrated above as equivalent to that in the cellular sample (ECM fraction) (Figure 2a~c). This (curve d) shows much smaller impedance. Together curves c and d suggest that the polyanionic HS strands on perlecan strongly repulse the electroactive reporter of EIS, [Fe(CN)6]3-/4-. After binding of the complex cation, this strong repulsion seems to be neutralized, and impedance comparable to curve e can be observed (curve e). After photo cleavage, a third curve similar to curve d and e can be observed (curve f), since the HS content has been largely removed from perlecan. In HCC, sulfatase 2 (sulf2), an HS 6-O-endo-sulfatases, is a major type of endosulfatase controlling the sulfation of tumor ECM HSPG14, 32, 33. Here, standard samples of sulf2 are incubated with the electrode, on which perlecan from the cellular sample (ECM fraction) is presented. The degree of sulfation is then examined using the complex cation (Figure 2d). Gradually decrease of signal readout is observed in proportion with the increase of enzyme concentration, indicating gradually lowered degree of sulfation due to the activity of the enzyme. The signal response also shows similar trend in relative to the acting time of the enzyme (Figure S6). On the other hand, the interactions between HS and various growth factor derived peptides are studied by the competitive binding of HS between the complex cation and the peptides. The mixtures of fixed amount of complex cation and gradually greater amount of peptide are incubated with the electrode presenting perlecan from the cellular sample (ECM fraction). The signal response (Figure 3a~d) represents the residual complex cation not displaced by the peptide. The peptides derived from the fibroblast growth factor 1 (FGF1), FGF2 and vascular epithelial growth factor (VEGF) are employed (Figure 3a~c, respectively). The former two peptides both exhibit binding strength to some extent inferior to the complex cation, with the third from VEGF slightly stronger than the cation. Using this VEGF peptide, competitive binding is repeated on the electrode presenting ECM perlecan pre-treated with gradu-

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Figure 1. Interactions between the peptide modified electrode and various standard and biological samples. (a) Electrochemical impedance spectra (EIS) recorded after binding of the peptide probe modified electrode with the recombinant core protein of perlecan of different concentrations. (b) Differential pulse voltammograms (DPVs) of HS cathodic stripping recorded after binding with purified HS, the electrode underwent the route a~c in Scheme 1. Inset shows the peak currents as a function of the concentration of the target. The error bars indicate standard deviation (n=3). (c) DPVs of HS cathodic stripping recorded after the incubation of serially diluted cellular samples (the ECM fraction). The electrode underwent the route a~f in Scheme 1. (d) EIS recorded after binding with recombinant core proteins of various HSPG. All targets are of 100 pM.

Figure 2. Study of HS sulfation using the complex cation. (a) DPVs of the complex cation recorded after incubation with the electrode on which is presented perlecan captured from the ECM fraction of the cellular sample (at 3.5×105cell/mL). Inset shows the peak currents as a function of the concentration of the complex cation. The error bars indicate standard deviation (n=3). (b) DPVs of HS cathodic stripping recorded following the route a~f in Scheme 1. The sample is the same as in (a). The meaning of data

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points and curves in the inset is the same as in (a). (c) EIS recorded at each step in the experimental treatments in (b). (d) DPVs of HS cathodic stripping recorded using the complex ion to interact with a peptide modified electrode which has previously been sequentially incubated with the same sample as in (a) and sulf2 of different concentrations as indicated on the graph. Inset is of similar meaning to those in (a) and (b).

Figure 3. Study of the interactions between the HS of perlecan and peptides derived from some growth factors, based on the competitive binding between the complex cation and the peptides with HS (route a~i in Scheme 1). (a)-(c) DPVs of HS cathodic stripping recorded after the competitive binding of the complex cation (10 µM) and different concentrations of (a) fibroblast growth factor 1 (FGF1), (b) FGF2 and (c) vascular epithelial growth factor (VEGF) peptides. Inset shows the peak currents as a function of peptide concentration. Error bars indicate the standard deviation (n=3). (d) The perlecan from the cellular sample is captured on the electrode surface, and subjected to the activity of different amount of sulf2. Then the electrode underwent the competitive binding between the complex cation and 10 µM VEGF derived peptide. Inset is of similar meaning to (a) ~ (c).

Figure 4. Study of the abundance of HS, sulfation of HS, as well as the interactions between HS and growth factor derived peptides, in cellular samples with suppressed sulf2 expression. Both the experimental and control groups have been primed with FGF2. (a) DPVs of HS cathodic stripping recorded following route a~c in Scheme 1. Inset is the cell viability. (b) DPVs recorded following route a~f in Scheme 1. Inset is the ratio between the corresponding peak currents in (b) and (a). (c) DPVs recorded following the route a~i in Scheme 1, using the FGF2 derived peptide.

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Figure 5. Box charts to show the distribution of the detected HS abundance (a) and HS sulfation (b) in paired tumoral and peritumoral samples of HCC, with the healthy liver as a control. The raw data is included as a column scatter plot to the left of each box. A curve corresponding to normal distribution is also displayed on top of the scatter plot. ally greater amount of sulf2 (Figure 43d). It can be seen that the signal response induced by the residual complex cation is gradually restored by the activity of sulf2, indicating that perlecan of lower level of sulfation can have decreased affinity towards the growth factor derived peptide. Based on these results, the sulfation of perlecan, and the consequently altered interaction with growth factor are studied in connection with the effect of sulf2 activity on the proliferation of the HepG2 cell. Sulf2 expression is knocked down in HepG2 primed with FGF2, and the FGF2 induced proliferation becomes inhibited, compared with the control of normal sulf2 expression (Figure 4a inset). The ECM perlecan is then studied with the above method (Figure 4). In Figure 1a, the response of HS does not differ evidently between the two cases, indicating similar amount of ECM perlecan. On the other hand, the response induced by the complex cation (Figure 4b) becomes higher with lowered sulf2 expression. It should be noted that this response does not directly represent the degree of sulfation. Both greater amount of perlecan and higher level of sulfation can contribute to this response, so the ratio between this and the former response is calculated (inset of Figure 4b). The normalized value can then represent the degree of sulfation, which is also higher with lowered sulf2 expression. Meanwhile, competitive binding experiments are conducted using the FGF2 derived peptide (Figure 4c), and the residual cation induced response also becomes lower, with lowered sulf2 expression, so that the bioavailibity of the growth factor becomes lower. These results may show the effectiveness of the proposed method in analyzing the bio-context relevant aspects of ECM enzyme activity in a tumor promoting microenvironment. The stability and reproducibility of the proposed method has also been examined and found to be acceptable (Figure S7), the standard deviation of all the repetitive measurements is less than 5%. The proposed method is then applied to clinical tissue samples of HCC, taken the non-fibrotic peritumoral tissue as the control. While the tissue samples from the healthy donors of liver transplant form the blank control. As is shown in Figure 5a, under the cancerous condition, HS become elevated in both the cancerous and paracancerous tissue, meanwhile as shown in Figure 5b, the sulfation is to some extent decreased in the cancerous samples. These observations, together with the above cellular experiments, may connect the activity of

sulfatase, as well as the sulfation of HS, to the progress of HCC. The pathology of HCC commonly results from chronic inflammatory conditions due to virus infection or the over uptake of alcohol 34-37. During this process, ECM components such as HS are constantly modified to adjust the ECM-cell interactions, driving the hepatocyte along a molecular cascade, finally leading to malignant transformation38-40. As we have observed (Figure 5), the abundance and sulfation of HS both have changes that may be connected with this pathological route. The increase of HS may point to the remodeling of ECM towards the formation of invasion front, while the lowered level of sulfation may facilitate cellular growth by releasing growth factors from the ECM reservoir. Conclusion In this work we have designed an assay for studying the sulfation and interactions of HS, as a means to evaluate the tumor promoting/inhibiting effect of sulfatase. An amyloid beta derived probe peptide is employed to detect ECM HS that exists in the form of HSPG. The affinity of the probe towards HSPG ensures high sensitivity in picomolar range. Taking advantage of the binding between HS and [Ru(bpy)2DPPZ]2+, this complex cation is employed to bind differentially with HS of different level of sulfation, and the subsequent photo-catalytic depolymerization of HS can generate amplified signal readout that can be related to the degree of HS sulfation. The activity of sulfatase 2, a major form of endo-sulfatase in HCC can be studied, in connection with the interactions between HS and various growth factors. These biochemical aspects of sulfatase 2 activity can be observed in parallel with the growth factor induced cell proliferation, and similar results can also be observed in analyzing clinical samples of HCC. These results may indicate the potential value of the proposed method in facilitating the diagnosis and prognosis of HCC in the future.

ASSOCIATED CONTENT Supporting Information Supporting material on the various experimental conditions, as wells as validation of detection principle, is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

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Corresponding Author * E-mail addresses: [email protected] (Y. Ding); [email protected] (Z. Wang); [email protected] (G. Li)

Author Contribution

ACKNOWLEDGMENT This work is supported by the National Natural Science Foundation of China (Grant Nos. 81501554, 21235003), and the Fundamental Research Funds for the Central Universities (Grant No. 021414380001).

REFERENCES 1. Buchheit, C. L.; Weigel, K. J.; Schafer, Z. T., Nat. Rev. Cancer 2014, 14., 632-641. 2. Gilkes, D. M.; Semenza, G. L.; Wirtz, D., Nat. Rev. Cancer 2014, 14., 430-439. 3. Belkin, A. M., Febs J. 2011, 278, 4704-4716. 4. Cabodi, S.; Camacho-Leal, M. d. P.; Di Stefano, P.; Defilippi, P., Nat. Rev. Cancer 2010, 10, 858-870. 5. Gialeli, C.; Theocharis, A. D.; Karamanos, N. K., Febs J. 2011, 278, 16-27. 6. Murphy, G.; Nagase, H., Febs J. 2011, 278, 2-15. 7. Nugent, M. A.; Zaia, J.; Spencer, J. L., Biochem.-Moscow 2013, 78, 726-735. 8. Uchimura, K.; Morimoto-Tomita, M.; Bistrup, A.; Li, J.; Lyon, M.; Gallagher, J.; Werb, Z.; Rosen, S. D., BMC Biochem. 2006, 7. 9. Gopal, G.; Shirley, S.; Raja, U. M.; Rajkumar, T., Asian Pac. J. Cancer Pre. 2012, 13., 641-646. 10. Hur, K.; Han, T.-S.; Jung, E.-J.; Yu, J.; Lee, H.-J.; Kim, W. H.; Goel, A.; Yang, H.-K., J. Pathol. 2012, 228., 88-98. 11. Lemjabbar-Alaoui, H.; van Zante, A.; Singer, M. S.; Xue, Q.; Wang, Y. Q.; Tsay, D.; He, B.; Jablons, D. M.; Rosen, S. D., Oncogene 2010, 29, 635-646. 12. Mondal, S.; Roy, D.; Pereira, J. C.; Chini, E. N.; Shridhar, V., Cancer Res 2014, 74. 13. Morimoto-Tomita, M.; Uchimura, K.; Bistrup, A.; Lum, D. H.; Egeblad, M.; Boudreau, N.; Werb, Z.; Rosen, S. D., Neoplasia 2005, 7, 1001-1010. 14. Yang, J. D.; Sun, Z.; Hu, C.; Lai, J.; Dove, R.; Nakamura, I.; Lee, J.-S.; Thorgeirsson, S. S.; Kang, K. J.; Chu, I.-S.; Roberts, L. R., Genes Chromosomes Cancer 2011, 50, 122-135. 15. Carnachan, S. M.; Bell, T. J.; Sims, I. M.; Smith, R. A. A.; Nurcombe, V.; Cool, S. M.; Hinkley, S. F. R., Carbohydr. Polym. 2016, 152, 592-597. 16. Galeotti, F.; Volpi, N., J. Chromatogr. A 2016, 1445, 68-79. 17. Ji, X. H.; Hu, G. X.; Zhang, Q. Y.; Wang, F. S.; Liu, C. H., Carbohydr. Polym. 2016, 152, 337-342. 18. Auray-Blais, C.; Lavoie, P.; Tomatsu, S.; Valayannopoulos, V.; Mitchell, J. J.; Raiman, J.; Beaudoin, M.; Maranda, B.; Clarke, J. T. R., Anal. Chim. Acta 2016, 936, 139-148. 19.Snow, A. D.; Kinsella, M. G.; Parks, E.; Sekiguchi, R. T.; Miller, J. D.; Kimata, K.; Wight, T. N., Arc. Biochem. Biophy. 1995, 320, 84-95. 20. Billings, P. C.; Pacifici, M., Connect. Tissue Res. 2015, 56, 272280. 21. Wong, P.; Hampton, B.; Szylobryt, E.; Gallagher, A. M.; Jaye, M.; Burgess, W. H., J. Biol. Chem. 1995, 270., 25805-25811. 22. Baird, A.; Schubert, D.; Ling, N.; Guillemin, R., Proc. Nat. Acad. Sci. U. S. A. 1988, 85, 2324-2328. 23. Yamazaki, Y.; Tokunaga, Y.; Takani, K.; Morita, T., Biochem. 2005, 44, 8858-8864. 24. Cheng, T.-T.; Yao, J.-L.; Gao, X.; Sun, W.; Shi, S.; Yao, T.-M., Analyst 2013, 138, 3483-3489.

Page 8 of 9

25. Piech, R.; Paczosa-Bator, B.; Golen, K., Inter. J. Electrochem. Sci. 2012, 7, 5122-5132. 26. Liu, X.-W.; Lu, J.-L.; Chen, Y.-D.; Li, L.; Zhang, D.-S., Inorg. Chim. Acta 2011, 379 (1), 1-6. 27. Liu, Z. C.; Perlin, A. S., Carbohydrate Res. 1994, 255, 183-191. 28. Li, G.; Cai, C.; Li, L.; Fu, L.; Chang, Y.; Zhang, F.; Toida, T.; Xue, C.; Linhardt, R. J., Anal. Chem. 2014, 86, 326-330. 29. Hartshorn, R. M.; Barton, J. K., J. Am. Chem. Soc. 1992, 114, 5919-5925. 30. Sarrazin, S.; Lamanna, W. C.; Esko, J. D., CSH Perspective Biol. 2011, 3. 31. Zhou, Z. J.; Wang, J. M.; Cao, R. H.; Morita, H.; Soininen, R.; Chan, K. M.; Liu, B.; Cao, Y. H.; Tryggvason, K., Cancer Res. 2004, 64, 4699-4702. 32. Peterson, S. M.; Iskenderian, A.; Cook, L.; Romashko, A.; Tobin, K.; Jones, M.; Norton, A.; Gomez-Yafal, A.; Heartlein, M. W.; Concino, M. F.; Liaw, L.; Martini, P. G. V., Bmc Cancer 2010, 10. 33. Lai, J.-P.; Oseini, A. M.; Moser, C. D.; Yu, C.; Elsawa, S. F.; Hu, C.; Nakamura, I.; Han, T.; Aderca, I.; Isomoto, H.; Garrity-Park, M. M.; Shire, A. M.; Li, J.; Sanderson, S. O.; Adjei, A. A.; FernandezZapico, M. E.; Roberts, L. R., Hepatology 2010, 52, 1680-1689. 34. Nguyen, A. H.; Berim, I. G.; Agrawal, D. K., Exp. Rev. Clin. Immunol. 2015, 11, 849-857. 35. Park, B.-J.; Lee, Y.-J.; Lee, H.-R., World J. Gastroenterol. 2014, 20, 2168-2175. 36.Peng, Z.; Li, C.-F.; Hao, J.-L.; Meng, S.-D., Pro. Biochem. Biophy. 2014, 41, 17-23. 37. Weber, A.; Boege, Y.; Reisinger, F.; Heikenwalder, M., Swiss Med. Wkly 2011, 141. 38. Dudas, J.; Ramadori, G.; Knittel, T.; Neubauer, K.; Raddatz, D.; Egedy, K.; Kovalszky, I., Biochem. J. 2000, 350, 245-251. 39. Khurana, A.; Beleford, D.; He, X.; Chien, J.; Shridhar, V., Am. J. Cancer Res. 2013, 3, 34-45. 40. Tatrai, P.; Egedi, K.; Somoracz, A.; van Kuppevelt, T. H.; ten Dam, G.; Lyon, M.; Deakin, J. A.; Kiss, A.; Schaff, Z.; Kovalszky, I., J. Histochem. Cytochem. 2010, 58, 429-441.

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Graphical Abstract 34x22mm (300 x 300 DPI)


Evaluating Tumor-Associated Activity of Extracellular Sulfatase by

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