Electrochemical Detection and Distribution Analysis of Î²-Catenin for

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Electrochemical detection and distribution analysis of #-catenin for the evaluation of invasion and metastasis in hepatocellular carcinoma Yue Yu, Hao Li, Luming Wei, Liudi Li, Yitao Ding, and Genxi Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00037 • Publication Date (Web): 04 Mar 2016 Downloaded from http://pubs.acs.org on March 6, 2016

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Analytical Chemistry

Electrochemical detection and distribution analysis of β-catenin for the evaluation of invasion and metastasis in hepatocellular carcinoma Yue Yu†#, Hao Li‡#, Luming Wei‡, Liudi Li‡, Yitao Ding†,* 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.

§

. Laboratory of Biosensing Technology, School of Life Sciences, Shanghai University, Shanghai 200444, China.

ABSTRACT: Pro-metastatic cell signaling controls the switch to distant metastasis and the final cancer death. In hepatocellular carcinoma (HCC), this death switch is turned on by the multi-protein interactions of β-catenin with many transcription factors, so a method to assay the bioactivity of β-catenin to participate in these pro-metastatic protein/protein interactions has been proposed in this work. This method employs cost-effective peptide-based protein targeting ligands, while the electrochemical catalytic crosslinking in this method also “finalize” the non-covalent molecular recognition, so that the robustness can be improved to enable detection of relatively more complex bio-samples. In studying clinical samples with the proposed method, the cellular distribution and overall expression of β-catenin show a parallel with the pathological grade of the sample, particularly, nuclear translocation. The pro-metastatic activation of β-catenin can also be observed as evidently correlated with higher-grade cases, suggesting the active role of β-catenin in promoting metastasis. According to these results, the proposed method may have the prospective use as a prognostic tool for evaluating the potential of invasion and metastasis in cancer.

Although it has been widely accepted that metastasis is the foremost cause of cancer death 1-4, relatively little is known about the signaling networks governing the timing, probability, and distribution of metastasis 5-8. Therefore, in order to improve the current prognostic tools and therapeutic strategies for metastatic cancer, it is of urgent consideration to learn more about the pro-metastatic signaling pathways 9-11, especially essential protein-protein interactions on the pathway 12, which may not only serve as functional protein-based pathway biomarkers for screening potential pro-metastatic signaling, but also can be the target of therapeutic intervention to check metastatic progression. Among many recently discovered and defined pro-metastatic pathways, Wnt signaling pathway is an important signaling shaft in the tumorigenesis and metastasis of hepatocellular carcinoma (HCC) 13-16, and Wnt signaling pathway has β-catenin as its primary intracellular effector. In the presence of Wnt signal, multi-protein interactions between β-catenin and BCL-9, TCF, as well as other cofactors, result in Wnt gene activation 17-19. Moreover, these β-catenin/protein interactions can indicate the nuclear translocation of β-catenin, a process closely associated with the acquiring of metastatic phenotype 20, because β-catenin is also a regulatory protein of cell-cell contact tethering cells to the surrounding microenvironment. Therefore, the detection of the pro-metastatic activity of β-catenin, and the translocation of β-catenin, can provide early warning of invasiveness and metastasis. Many protein/protein interactions are realized through the interaction between pockets, grooves or other binding motifs

of the effector protein and peptide strands from the corresponding cofactor proteins 21. In the case of β-catenin, two αhelix peptides from BCL-9 and TCF-4 can simultaneously bind with their respective groove-like binding sites on βcatenin22, and trigger the downstream Wnt gene activation 23. Protein fragments and functional motifs have already been employed in the detection of β-catenin24-26, but the direct application of peptide has not to be attempted. So, we propose in this work that these peptides may be elaborately designed as probes to detect the above pro-metastatic β-catenin/cofactor interactions, thus the nuclear translocation of β-catenin can be evaluated based on the detected interactions. On the other hand, to use these peptide-based probes in clinical samples, interference from non-specific absorption should be minimized. Usually, in an interface assay using such probes, the interference in standard sample or mixed sample can be reduced with relatively mild rinsing, while violent rinsing with denaturant is required for complicated biological samples. However, the rinsing with denaturant is too violent for the peptide/β-catenin interactions to endure, since the complex formed by the probes and β-catenin may dissolve as the result of protein denaturation. So we have also included clickable groups in the peptide probes to induce cross-linking between the two probes (Scheme 1a), so as to withstand thorough rinsing indispensible for reliable analytical performance in detecting clinical samples. To quantitatively detect the β-catenin activity, the molecular recognition of β-catenin by the two probes is employed to control the kinetics of cross-linking from two aspects (Scheme 1a~c). First, simultaneous binding

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Scheme 1. (a) Design of the two clickable peptide-based probes. Different functional motifs in the probes are represented with different colors. The violet sequence in the biosensing probe and the blue sequence in the signaling probe are the peptide motifs recognizing the target protein. (b) The assay established using the two probes. (c) Detailed schemes of chemical reactions in the second and third step of (b), the left scheme is corresponding to the second step in (b), while the right scheme shows the reaction in the third step in (b).

of the two peptide probes can bring them into vicinity to facilitate their cross-linking (Scheme 1b). Second, Cu (I) ion, the catalyst required for cross-linking, is incorporated into one of the probes in its non-active Cu (II) form (Scheme 1a). So, for an interface assay on the surface of the electrode, the valency of the catalyst, as well as its activity, can be electrochemically controlled to realize quantitative cross-linking of the β-catenin recognized probes (Scheme 1b), giving rise to signal readout proportional to the abundance of β-catenin. Besides, the Cu (I/II) ion, as the catalyst, can also catalyze oxidative generation of electro-active species (Scheme 1c), so the final signal readout can also be effectively amplified. Experimental Section Chemicals and biological samples The clickable peptide probes, their sequence as shown in Scheme 1a, were synthesized by Shanghai Science Peptide Biological Technology Co., Ltd, as lyophilized powder, purity>95%. Human recombinant β-catenin was from Sigma-Aldrich. Other reagents were of analytical-grade. Powder of the peptide probe was dissolved in 10 mM phosphate buffer solution (PBS) (pH 7.4) to the con-

centration needed. The same solution was used to dissolve βcatenin. For all solutions, double-distilled water (ddH2O) was prepared in Milli-Q purification system to 18 MΩ·cm. HA22T cell line was cultured in Dulbecoo’s Modified Eagle’s Medium (DMEM, from Gibco co.) containing 10% fetal cattle serum (FCS, from Hyclone co.) and maintained in a humidified atmosphere with 5% CO2 at 37 °C, cell line Hep3 was cultured in Basal Medium eagle (BME, from Gibco co.), the culture had also been supplemented with 10% fetal cattle serum and maintained in a humidified atmosphere with 5% CO2 at 37 °C. For β-catenin detection, the cells were collected, counted and diluted to 1×105 cell/mL, followed by fractioning with a nuclear fraction kit (Abnova), both nuclear and cytoplasmic fractions were reserved for detection. Biopsy samples from hepatocellular carcinoma patients were obtained from the Department of General Surgery of the Affiliated Hospital of Nanjing University, after elected consent by the local ethical committee. 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 pellet was re-suspended with 10 mM PBS and fractioned with a nuclear fraction kit (Abnova), following the instruction of the manufacturer. The nuclear and cytoplas-

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Figure 1. Validation of the design principle, the electrode has undergone the steps of β-catenin detection described in the Experimental Section, concentration of target protein: 10 nM. (a) Step-wise electrochemical impedance spectra of the proposed biosensing procedure. The curves are respectively corresponding to a, bare electrode, b, biosensing probe modified electrode, c, the electrode with captured target protein and the signaling probe, d, the electrode after cross-linking, and e, the electrode after thorough rinsing. (b) Square wave voltammograms (SWVs) of DAP showing the effect of violent rinsing in the presence (the red curve)/absence (the black curve) of cross-linking step. mic fractions were then used as the targets in the correspondthen transferred into a 1 mM HCl solution (1 mL) containing ing measurements of nucleic and cytoplasmic beta-catenin. 0.1 mg/ml ο-phenylenediamine (OPD). The solution was then placed in 50 °C water bath for 30 min. After being cooled to ITC study of probe/Smo interaction Isothermal titration room temperature, this solution was buffered with 4 mL 10 calorimetry (ITC) measurements were conducted using a MimM PBS pH 7.4. Signal response of the generated 2,3croCal ITC200 System (GE healthcare life sciences) at 25 °C. diaminophenazine (DPA) was then recorded in the buffered The titration schedule consisted of 38 consecutive injections of reaction solution. 1 µL with at least a 120 s interval between injections. All solutions were degassed prior to titration. Electrochemical measurements These steps were essentially the same as previously reported 27. Briefly, electrochemical Electrode treatment and modification These steps were 27 measurements were carried out on a CHI660D Potentiostat essentially the same as previously reported . Firstly, gold (CH Instruments) with a conventional three-electrode system: disk electrode (3 mm diameter) was cleaned using piranha the electrode immobilized with peptide as the working elecsolution (70% concentrated sulfuric acid, 30% H2O2) for 5 min trode, a saturated calomel electrode (SCE) as the reference (Caution: Piranha solution reacts violently with organic solelectrode and a platinum wire as the counter electrode. Square vents and should be handled with great care!). After that, the wave voltammograms (SWVs) were recorded in 10 mM PBS, electrode was rinsed with double-distilled water. The electrode pH 7.4, which was deoxygenated by purging with nitrogen gas was then polished with 1 µm, 0.3 µm alumina slurry in seand maintained under this inert atmosphere during the electroquence. Subsequently, ultrasonicating in both ethanol and chemical measurements. water was used to remove residual alumina powder. Finally, the electrode was immersed in nitric acid (50%) for 30 min, Results and Discussion followed by electrochemically cleaning with 0.5 M H2SO4 to The proposed method to assay β-catenin activity has been remove remaining impurities.After being dried with nitrogen, illustrated in Scheme 1. Specifically, the two peptides are dethe electrode was immerged in 50 µL assembly solution (2.5 signed as the biosensing probe, and the signal probe contains a µM bi-functional peptide and 5 mM TCEP in 10 mM PBS, pH trimeric glycylhistidyllysine (GHK) motif to complex with Cu 7.4) for 16 h at 4 °C, TCEP was used to prevent disulphide (II) (Scheme 1a). In the assay procedure (Scheme 1b), the formation between peptides. Then, the electrode was soaked in biosensing probe immobilized on the electrode surface cap100 µL MNH solution (1 mM MNH in 10 mM PBS, pH 7.4) tures the target β-catenin, while the signal probe can simultafor 3 h at room temperature. Finally, MNH non-specifically neously bind with β-catenin. Subsequent cyclic voltammetric adsorbed on the electrode surface was removed by thorough scans can reduce the Cu (II) coordinated by the signal probe to rinsing of the modified electrode, which was then dried under the active Cu (I) to catalyze cross-linking between the two mild nitrogen stream. closely-located probes. i.e. Cu (II) is electrochemically reduced to Cu (I), which is oxidized backed to Cu (II) while β-catenin detection The standard, fractioned cell or clinical acting as the catalyst of azide-alkyne click-coupling of the two samples were diluted at 1:10 with 100 µM signal probe, the probe; and the resulted Cu (II) can then be electrochemically mixture was immediately incubated with the sensing probe modified electrode at ambient temperature for 60 min, folreduced to generate once again the Cu (I) catalyst (Scheme 1b, lowed by cyclic voltammetric scanning over the potential c). Thorough rinsing is then applied, and after further supplerange of 0.6 V ~ 0 V for roughly 10 min at 1 V/s. After that, mentary incubation with Cu (II) ion (More Cu (II) is added to the electrode surface underwent repetitive rinsing with SDS. make sure that probes are complexed with Cu (II).), the crossOnce again the electrode was incubated briefly with 120 µM linked signal probes can be used for signal amplification, Cu(Cl)2, after gentle rinsing with ddH2O, the electrode was

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Figure 2. Quantitative analysis of β-catenin using the clickable peptide-based bioassay. The electrode has undergone the steps of βcatenin detection described in the Method section. Briefly, the biosensing probe modified electrode is incubated with the target and signaling probe, followed by electrochemical cross-linking step and thorough SDS rinsing, then after supplementary incubation with Cu (II), the electrode is employed to catalyze oxidative generation of DAP as the step of signal amplification. SWV of DAP is then recorded. (a) SWVs of 2,3-diaminophenazine (DAP) showing the gradual increase of signal response with β-catenin concentration. (b) Peak currents in (a) plotted as a function of β-catenin concentration. Inset is the linear range and the corresponding formula obtained via regression analysis. The error bars represent standard deviation from average (n=3). which involves using the above Cu (II/I) catalyst to catalyze the oxidation of ο-phenylenediamine (OPD) by dissolved oxygen, the product, electroactive 2,3-diaminophenazine (DAP) can then generate amplified signal (Scheme 1b, c). To realize the clickable peptide-based assay depicted above, the function of these peptide probes has to be investigated first. The binding of the two probes respectively with the target protein (Figure S1 (1) and (2)), as well as in the presence of each other (Figure S1 (1’) and (2’)), have been studied using isothermal calorimetry. From the resulting binding curves, conclusions can be drawn that binding of one probe does not interfere with the binding of the other, consistent with the previous report using the western blotting method 23. The proper functioning of these probes on the electrode surface is then studied using the electrochemical response of cupric ion complexed by the signal probe (Figure S2). As is shown in Figure S2a, the simultaneous interface and insolution recognition of the target protein by the biosensing probe and the signal probe can result in cupric ion response proportional to protein concentration. As a control, the sequential recognition first by the sensing probe and then by the signal probe can result in a similar response (Figure S2b), so the simultaneous recognition is selected for the subsequent experiments for a shorter assay time. If the sequences of the two probes are exchanged, the overall responses at all target concentrations are proportionally smaller than the original pair of probe sequence (Figure S2c), which may be due to lowered surface density of the probe, as well as diminished efficiency of target binding that can be achieved by the alternative arrangement, so the original arrangement is retained for the following experiments. Meanwhile, step-wise impedance measurement has also been conducted to validate the proposed detection procedure (Figure 1a). The bare electrode appears as a straight line (curve a), while surface modification with peptide results in a slight increase of impedance (curve b). Subsequent capturing of target results in evident increase of impedance, due to the presence of target protein on the surface (curve c). After the

cross-linking step, the impedance increases slightly (curve d), which may be due to slight disturbing of surface packing by the electrochemical scanning step involved in the cross-linking. After thorough rinsing with surfactant, only the cross-linked probes can be retained on the electrode surface, leading to large decrease of impedance (curve e). As a control, the effect of violent rinsing in the presence/absence of cross-linking pretreatment has been compared (Figure 1b). This result shows that covalent coupling between the two probes can ensure their retention; while the proteins may be de-natured by the violent detergent rinsing and removed from the surface, but the molecular recognition of the target protein by the two probes are finalized by cross-linking of the probes and their retention on the surface. The electrochemical control of probe cross-linking is then studied, still using the response of complexed cupric ion. As can be seen from Figure S3 and Figure S4, after the initial increase of response with electrochemical treatment, both the voltammetric scans over large potential ranges and prolonged electrochemical scanning can result in a second phase of much slower increment of responses. This may be owing to the

Figure 3. SWVs of DAP showing the specificity of the assay. All species are of 10 nM. The electrode has undergone the steps of β-catenin detection the same as in Figure 2.

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Figure 4. SWVs of DAP showing the detected cellular distribution of β-catenin in two cell lines, the red curves are corresponding to the cell line of higher invasive power (cell concentration for sample preparation: 1×105 cell/mL). The cellular sample is collected and fractioned as described in the Experimental Section, and the electrode has undergone the steps of βcatenin detection the same as in Figure 2. surplus in-solution signal probes beginning to be transferred to the electrode and cross-linked, as more and more target-bound signal probes on the interface gradually become cross-linked. Therefore, relatively mild conditions of electrochemical treatment, on the border of the two phases, are selected to obtain an amount of surface cross-linked signal as large as possible, at the same time without evident false-positives. Various experimental steps have been optimized. First, as is shown in Figure S5, the cross-linking step can offer a satisfactory analytical performance, compared with the control without cross-linking. Then, the incubation time for the recognition of target protein by the two probes has been studied (Figure S6), and 60 min is selected as the proper incubation time. The concentration of cupric ion for supplementary incubation with the surface cross-linked signal probes has also been optimized, yielding 120 µM as the optimal concentration (Figure S7). Lastly, the amount of reactant used in the final step of catalytic generation of electroactive species has been studied. Experimental results reveal

that the reaction catalyzed by surface complexed cupric ion can approach saturation with 400 µg/mL reactant (Figure S8), so a slightly surplus amount of reactant at 1 mg/mL has been chosen. Under these optimized conditions, the obtained signal response can show a proportional increase at a large range of target concentrations, in detecting standard samples containing β-catenin. Specifically, a linear range between signal response and the logarithm of target concentration can be established from 32 pM to 10 nM (Figure 2), with a limit of detection calculated at a signal-to-noise ratio of 3:1 of less than 10 pM. Also, the receptivity of measurement can be acceptable since all standard deviation of independent repetitive measurements are within 5% (n=3). The specificity of this assay has also been studied with several control species, all of which can only have a background level of response (Figure 3). Particularly, histone, the most abundant protein in the nucleus where pro-metastatic β-catenin is also present, can have no evident interference to the detection of β-catenin, confirming the possibility of using this method to detect the translocated βcatenin. The translocation of β-catenin is an evident sign of invasiveness and metastasis. To promote invasion and metastasis, β-catenin, ceased to be a cytoplasm deviled regulatory protein of the adherin-linked cell-cell contact, is sequestered from the inner surface of the cell membrane, and translocated into the nucleus to form large transcription activation complexes with BCL-9, TCF and other cofactors. Therefore, the re-distribution of β-catenin between cytoplasm and nuclei can indicate prometastatic activation of β-catenin. To this end, the distribution of β-catenin in the cytoplasm and nuclei of two cell lines with different invasive power is compared. As is shown in Figure 4, the more invasive cell line has proportionally more β-catenin translocated inside the nucleus. This study of β-catenin cellular distribution is further extended to clinical samples of HCC, since β-catenin has recently been found as a biomarker of metastasis for HCC 28 due to its active role in the up-regulation of Wnt signaling and remodeling of cellular skeleton as well as cell-cell contact. Accumulating data suggest that Wnt signaling is one important

Figure 5. Box chart to show the cellular distribution of the detected concentration of β-catenin in patients of HCC, grouped by the pathological grade. For each sample, the detected β-catenin in cancerous tissue is normalized as the fold of adjacent normal tissue. Each box includes the maximum, minimum, mean marked on the graph, in addition to the 25th, median, and 75th percentiles. 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. (b) Comparison between our method and commercially available ELISA method in detecting the above clinical samples.

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component of the switch to metastasis in the cellular signaling network10, 29-32. At the same time, as the nuclear effector of Wnt signaling, β-catenin also connects this signaling to the early steps of metastasis, the remodeling of surrounding tissues and the forming of the front edge of invasion. All these upstream and downstream events can have the nuclear translocation of β-catenin as its pivotal phase. So we examine βcatenin in clinical samples to reveal whether the re-distribution of β-catenin towards pro-metastatic geno- and phenotypic changes can have any correlation with the advancing of HCC. Based on pathological examination, clinical samples of HCC tissue have been grouped according to their pathological grades (I ~ III), and the nuclear and cytoplasmic fraction prepared from the samples are assayed with our proposed method for the abundance of β-catenin. As is shown in Figure 5, with higher grades, the nuclear translocation becomes more prominent, and for grade III, the nuclear fraction dominates the detected β-catenin (Figure 5a~c). It can also be noted that the overall abundance of β-catenin has also elevated with the grade of HCC. Based on these results, it might be safe to suggest that the nuclear translocation of β-catenin, to some extent its overall expression as well, can be a preliminary prognostic marker for the potential of invasiveness and metastasis in HCC. The commercially available ELISA kit can generate a result comparable with that using our method (Figure 5b), although the limit of detection in the range of 0.1 nM is slightly inferior to our method. Conclusion In summary, a method to assay the ability of β-catenin to participate in pro-metastatic cell signaling has been proposed in this work. Pro-metastatic cell signaling controls the switch to distant metastasis and the final cancer death. In hepatocellular carcinoma (HCC), this death switch is turned on by the multi-protein interactions of β-catenin with many transcription factors. In light of these interactions, two peptide-based probes, which are derived from the molecular partners of β-catenin involved in these protein interactions, are designed. Moreover, clickable groups are also designed to be incorporated into the probes to enable an electrochemically controlled catalytic cross-linking between the two probes. Therefore, this method may enable bioanalysis of clinical samples of HCC. In studying clinical samples with the proposed method, the cellular distribution and overall expression of β-catenin show a parallel with the pathological grade of the sample, particularly, nuclear translocation. The pro-metastatic activation of β-catenin can also be observed as evidently correlated with higher-grade cases, suggesting the active role of β-catenin in promoting metastasis.

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

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Author Contribution #

Y. Yu and H. Li: These authors contribute equally to this work.

ACKNOWLEDGMENT This work is supported by the National Natural Science Foundation of China (Grant Nos. 81501554, 21235003, 21327902, J1103512, J1210026).

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Scheme 1. (a) Design of the two clickable peptide-based probes. Different functional motifs in the probes are represented with different colors. The violet sequence in the biosensing probe and the blue sequence in the signaling probe are the peptide motifs recognizing the target protein. (b) The assay established using the two probes. (c) Detailed schemes of chemical reactions in the second and third step of (b), the left scheme is corresponding to the second step in (b), while the right scheme shows the reaction in the third step in (b). 140x135mm (300 x 300 DPI)



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Figure 1. Validation of the design principle, the electrode has undergone the steps of β-catenin detection described in the Experimental Section, concentration of target protein: 10 nM. (a) Step-wise electrochemical impedance spectra of the proposed biosensing procedure. The curves are respectively corresponding to a, bare electrode, b, biosensing probe modified electrode, c, the electrode with captured target protein and the signaling probe, d, the electrode after cross-linking, and e, the electrode after thorough rinsing. (b) Square wave voltammograms (SWVs) of DAP showing the effect of violent rinsing in the presence (the red curve)/absence (the black curve) of cross-linking step. 56x23mm (300 x 300 DPI)


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Figure 2. Quantitative analysis of β-catenin using the clickable peptide-based bioassay. The electrode has undergone the steps of β-catenin detection described in the Method section. Briefly, the biosensing probe modified electrode is incubated with the target and signaling probe, followed by electrochemical crosslinking step and thorough SDS rinsing, then after supplementary incubation with Cu (II), the electrode is employed to catalyze oxidative generation of DAP as the step of signal amplification. SWV of DAP is then recorded. (a) SWVs of 2,3-diaminophenazine (DAP) showing the gradual increase of signal response with βcatenin concentration. (b) Peak currents in (a) plotted as a function of β-catenin concentration. Inset is the linear range and the corresponding formula obtained via regression analysis. The error bars represent standard deviation from average (n=3). 54x22mm (600 x 600 DPI)



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Figure 3. SWVs of DAP showing the specificity of the as-say. All species are of 10 nM. The electrode has undergone the steps of β-catenin detection the same as in Figure 2. 54x41mm (600 x 600 DPI)


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Figure 5. Box chart to show the cellular distribution of the detected concentration of β-catenin in patients of HCC, grouped by the pathological grade. For each sample, the detected β-catenin in cancerous tissue is normalized as the fold of adjacent normal tissue. Each box includes the maximum, minimum, mean marked on the graph, in addition to the 25th, median, and 75th percentiles. 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. (b) Comparison between our method and commercially available ELISA method in detecting the above clinical samples. 51x21mm (300 x 300 DPI)


Electrochemical Detection and Distribution Analysis of Î²-Catenin for

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