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Cite this: Phys. Chem. Chem. Phys., 2014, 16, 5276
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Modulation of glyceraldehyde-3-phosphate dehydrogenase activity by surface functionalized quantum dots† Srabanti Ghosh,*a Manju Ray,b Mahua Rani Das,c Adrita Chakrabarti,a Ali Hossain Khan,a D. D. Sarmad and Somobrata Acharya*a Enzymatic regulation is a fast and reliable diagnosis tool via identification and design of inhibitors for modulation of enzyme function. Previous reports on quantum dots (QDs)–enzyme interactions reveal a protein-surface recognition ability leading to promising applications in protein stabilization, protein delivery, bio-sensing and detection. However, the direct use of QDs to control enzyme inhibition has never been revealed to date. Here we show that a series of biocompatible surface-functionalized metal–chalcogenide QDs can be used as potent inhibitors for malignant cells through the modulation of enzyme activity, while normal cells remain unaffected. The in vitro activity of glyceraldehyde-3phosphate dehydrogenase (GAPDH), an enzyme involved critically in the glycolysis of cancer cells, is
Received 16th August 2013, Accepted 10th December 2013
inactivated selectively in a controlled way by the QDs at a significantly low concentration (nM).
DOI: 10.1039/c3cp53489h
mechanisms owing to the site-specific interactions, enabling control over the inhibition kinetics. These complementary loss-of-function probes may offer a novel route for rapid clinical diagnosis of malignant
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cells and biomedical applications.
Cumulative kinetic studies delineate that the QDs undergo both reversible and irreversible inhibition
1. Introduction The emergence of the design of enzyme modulators provides a potent tool to target malignant cells via blocking an enzyme activity that influences metabolic imbalance.1 The Warburg effect, which explains the increased glycolytic capacity of cancer cells, is the most fundamental metabolic alteration during malignant transformation.2 The increased glycolytic capacity of a cancer cell is associated with mitochondrial defects and the abnormal expression of metabolic enzymes.3 Glyceraldehyde-3phosphate dehydrogenase (GAPDH) is one of the key enzymes in the glycolytic cascade, which constitute a novel target for anticancer therapy due to overexpression in many cancer tissues such as lung cancer, prostatic adenocarcinoma, renal cell carcinoma, breast carcinoma cells, hepatocellular carcinoma etc. without affecting their normal counterparts.4–6 Normal cells adapt a
Centre for Advanced Materials, Indian Association for the Cultivation of Science, Kolkata 700032, India. E-mail: [email protected], [email protected] b Division of Molecular Medicine, Bose Institute, Kolkata 700 054, India c Biological Chemistry, Indian Association for the Cultivation of Science, Kolkata-700032, India d Solid State and Structural Chemistry Unit & Centre for Condensed Matter Theory Indian Institute of Science, Bangalore 560012, India † Electronic supplementary information (ESI) available: Additional discussions and TEM images. See DOI: 10.1039/c3cp53489h
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alternative metabolic pathways to sustain their energy requirements during increased glycolytic activity during which ATP generation through mitochondrial oxidative phosphorylation is compromised. Consequently, frequent mitochondrial DNA mutations, malfunction of the electron transport chains, aberrant enzyme expression in energy metabolism and insufficient oxygen availability in the cellular microenvironment are generally evidenced in a variety of human cancers.7 The inner core tumor cells naturally become hypersensitive to materials as a result of abnormal cellular bioenergetics, which block the glycolytic pathways at different steps. Hence, the development of novel glycolytic inhibitors can be utilized as a new class of cancer biomarkers and in a variety of therapeutic applications.8 GAPDH inhibitors can block the conversion of the oxidative phosphorylation of glyceraldehyde 3-phosphate (GAP) to 1,3-bisphosphoglycerate in the sixth step of glycolysis, which is effective in the inhibition of cellular respiration causing cancer cell death.9,10 Thus, the glycolytic pathway provides a biochemical basis for novel therapeutic strategies to prevent cancer cell growth by the pharmacological inhibition of glycolysis. Several molecules exhibit promising anticancer activities as single units or in combination with other therapeutic modalities based on enzyme inhibition to influence different cellular processes such as apoptosis and angiogenesis.11,12 Unfortunately, these molecular inhibitors generally do not show
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specificity for the inhibition of enzyme activity.13 Thus, an alternative strategy to achieve a target-specific agent with efficient therapeutic selectivity that differentiates between normal and cancer cells in course of biochemical metabolism is crucial. Several studies have suggested that the surface modified quantum dots (QDs) exhibit improved binding with enzyme molecules which may allow a better understanding of the underlying kinetic mechanisms at the nanoparticle interface for designing QDs based biological tools.14,15 Recently, the use of QDs together with bio-molecules as diagnostic tools has shown major advantages over traditional chemotherapeutic compounds in cancer research.16,17 However, the use of QD often leads to aggregation upon conjugation with protein, limiting the effectiveness of disease diagnosis.18 Here we report on the use of metal chalcogenide QDs as potential enzyme inhibitor with differential enzymatic regulation. In vitro inhibition studies of GAPDH in presence of QDs suggest that the binding of QDs to the enzyme molecules slows down the rate of the enzyme catalyzed reaction probing QDs as a potential enzyme inhibitor. We investigated the mechanistic pathways for enzyme inhibition using GAPDH from normal and tumour associated mammalian tissues, respectively. In vitro enzyme kinetic analyses show that QDs have a marked affinity towards enzyme molecules in comparison to the substrates following a differential modulation of enzyme activities via reversible or irreversible targeted inhibition. Spectroscopic evidence reveals that the binding of QDs with GAPDH retains the protein structure both for normal and tumour associated tissues. Our results highlight new insights into the underlying correlation between the antiproliferative activity and inhibition of enzyme activity by QDs in cancer cells without alteration of the structural conformation.
2. Experimental 2.1
Reagents
3-Mercaptopropionic acid, L-cysteine hydrochloride, cadmium chloride hexahydrate, glycine, magnesium chloride, sodium hydroxide, ethylenediaminetetraacetic acid (EDTA), p-nitrophenyl phosphate, sodium chloride (NaCl), potassium chloride (KCl) and disodium hydrogen phosphate (Na2HPO4) were purchased from Merck, Germany. N-Acetyl-L-cysteine (NAC), tellurium powder, sodium borohydrate (NaBH4), triethylamine hydrochloride, glyceraldehyde-3-phosphate (GAP), nicotinamide adenine dinucleotide (NAD), b-mercaptoethanol, glycerol, glyceraldehyde-3phosphate dehydrogenase (GAPDH) from rabbit skeletal muscle, 3-methylcholanthrene, a-oxoglutarate, ADP (disodium salt), succinate, malonate, and pyridoxal 5 0 -phosphate (PP) were obtained from Sigma Chemical Co., St. Louis, MO, USA. Sephadex G-50 was purchased from Calbiochem and Pharmacia Fine Chemicals, Sweden. All the chemicals were of analytical grade or highest purity available and were used as-obtained. Milli-Q water (Millipore) was used as a solvent. 2.2
Synthesis of thiol-capped quantum dots
CdS and CdTe QDs were synthesized following the method as reported earlier with trivial modification.19,20 In a typical synthesis,
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an aqueous solution of Cd2+ ion (2 102 M) is dissolved in 100 mL water and 5 102 M of the thiol stabilizer, such as 3-mercapto propionic acid (MPA), N-acetyl-L-cysteine (NAC), cysteine hydrochloride (Cys) used as capping agents followed by adjusting pH 11 with 1 M NaOH solution. Then a freshly prepared aqueous solution of H2S of known concentration (estimated iodometrically using sodium arsenite) was added in a stoichiometric amount to the N2 purged Cd–thiolate complex solution. For CdTe QDs, a freshly prepared aqueous solution of sodium hydrogen telluride (NaHTe) was added to maintain the final molar ratio of Cd : thiols : Te – 1 : 2 : 0.5. The resultant solution was refluxed at 100 1C and aliquots were collected at various time intervals having different sizes of QDs. To remove the excess Cd–thiolate complexes at the end of the synthesis, 2-propanol was added to the reaction mixture to precipitate CdTe and CdS QDs. The as-prepared product was freeze-dried under vacuum for further experiments. 2.3
GAPDH assay
GAPDH activity was measured spectrophotometrically by monitoring the reduction of NAD+ to NADH at 340 nm. The reaction was carried out at 25 1C and was initiated by the addition of 1–5 mg of the enzyme to the reaction mixture containing 50 mmol triethanolamine buffers, 50 mmol Na2HPO4, 0.2 mmol EDTA, 1 mmol NAD and 0.5 mmol of GAP. To monitor the reaction, the increase in absorbance at 340 nm due to the formation of NADH from NAD was noted at 30 second intervals, the values remaining almost linear for 5 minutes. One unit of activity is defined as the amount of enzyme required to convert 1 mmol of NAD+ to NADH per minute at standard assay conditions (pH 8.5, 25 1C). 2.4
Development of sarcoma in mice
Animal experiments were carried out in accordance with the guidelines of the institutional ethics committee (IEC). Appropriate precautions were taken to minimize pain or discomfort to animals. 2.5
Development of tumour
Sarcoma tissue was developed in the left hind leg of mice by intramuscular injection of 3-methyl-cholanthrene. The carcinogen was dissolved in olive oil by placing it in warm water-bath and 0.1 mL of olive oil containing 0.2 mg of the carcinogen was injected into each mouse thrice with a one-week interval. After 12–14 weeks a full-grown tumour was developed. The malignancy was confirmed by histological examination where differentiated muscle cells are conspicuously visible in normal mouse muscle, and highly differentiated sarcoma tissue was observed where tumour was developed by 3-methyl-cholanthrene. 2.6 Purification of GAPDH from normal muscle, sarcoma tissue and Ehrlich ascites carcinoma (EAC) cells 15 gm of either normal mice muscle or sarcoma tissue was homogenized in an Omni GLH International homogenizer with four volumes of 50 mM triethanolamine-HCl buffer, pH 7.4 containing 10 mM EDTA and 10 mM b-mercaptoethanol. The tissue and the buffer were pre-cooled and all operations were carried out at 0–4 1C. From the homogenate, GAPDH was
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purified by gel filtration and ion exchange chromatography. The enzyme fractions after the DEAE-sephacel column step were purified 28 and 96 fold for normal muscle and sarcoma tissues, respectively. The specific activities of GAPDH at this stage were 112 for normal muscle and 313 for sarcoma tissue.
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2.7
Cell lines
Sarcoma 180 cells and EAC cells were maintained in the peritoneal cavity of Swiss albino female mice by weekly inoculation of 2 106 cells. 2.8
Cytotoxicity assays (MTT assay)
A murine cell line was used to determine the cytotoxicity of QDs. Cell toxicity was determined using the MTT assay, based on the reduction of a soluble tetrazolium salt by mitochondrial dehydrogenase of the viable cells to a water-insoluble coloured product (i.e. formazan).21 The activity of the enzyme and the amount of formazan produced is proportional to the number of cells alive. Briefly, C2C12 myoblast mice cells were seeded at a density of 2 105 cells per well in a 96-well microtiter plate for 18–24 h before the assay. The cells were incubated for 24 h at 37 1C under 5% CO2 in the presence of a varying concentration of quantum dots. Then, 50 ml of a 5 mg ml1 MTT (Sigma; St Louis, MO) was added to each well, followed by 4 h of incubation at 37 1C. The formazan crystals were then solubilized in 200 ml DMSO. The optical density (OD) at 570 nm was measured using an automated BioTeks Elisa Reader. The number of surviving cells were expressed as percentage viability = (the absorbance of the treated cells) background/the absorbance of the control (untreated cells) background 100. The cell toxicity of the extracted contents from scaffolds was rated as follows: severe (o30%); moderate (30–60%); slight (60–90%); or non-cytotoxic (490%) of MTT activity, compared to the control cells cultured in an extract-free medium. 2.9
Trypan blue exclusion assay
To measure the viable cell percentage, trypan blue dye was employed to stain the cells which did not have an intact membrane. The viable cells exclude the dyes and are not stained, which is a standard method to detect cell death. First, EAC and Sarcoma 180 cells were washed with PBS buffer pH 7.4 and then the cells were incubated for 1 h at 37 1C in the presence of varying concentrations of quantum dots. Finally, 10 mL of the trypan blue dye was added to each cellular suspension. The viable cell was counted through a hemocytometer, and the viability values were derived by comparing the samples with the negative control. The morphological changes of the C2C12 myoblast mice cells and malignant cells treated with QDs were visualized with a BX51WI fluorescence microscope (Olympus) equipped with 460–490 nm excitation filter setting and a DP71 digital camera and DP-BSW software for image acquisition. 2.10 Preparation of mitochondria and measurement of mitochondrial respiration Sarcoma tissue was collected and washed in a buffer containing 250 mM sucrose, 1 mM EDTA, 10 mM Tris/HCl pH 7.4 and
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0.1% BSA. After finely mincing the tissue, it was homogenized in 6 vol. of the buffer with 10 up and down strokes of a Potter– Elvehjem homogenizer and centrifuged at 1500g for 5 min. The supernatant was collected and centrifuged at 8000g for 15 min. The pellet was suspended and washed twice with same buffer by centrifuging at 8000g for 15 min and finally suspended in a minimum volume of the buffer. Additionally, mitochondria from EAC cells were prepared essentially by digitonin permeabilization by the method of Moreadith and Fiskum with some minor modifications.22 Mitochondrial oxygen consumption was measured with a Hansatech oxygraph fitted with a Clark electrode.23 The respiratory medium in a total volume of 2 mL contained 125 mM KCl, 10 mM Tris/MOPS (pH 7.4), 1 mM KH2PO4, 1 mM MgCl2, 1 mM EGTA and the respiratory substrates, which were usually 10 mM a-oxoglutarate or 10 mM pyruvate plus 10 mM malonate or 5 mM succinate. The mitochondrial protein in the medium was 0.3–0.6 mg. 2.11
Structural analysis
Circular dichroism (CD) spectra have been recorded by JASCO, CD Spectrometer; model J-815-150S using a 0.1 cm path length quartz cell in a wavelength range between 190 and 250 nm. The results were expressed as molar ellipticity [y] in units of m degree g cm2 dmol1. Molar ellipticity values were obtained using the relation: y = (Myobs)/10clnr where yobs is the observed ellipticity in degrees at a given wavelength, c is the protein concentration in mole per c.c. and l is the length of the light path in cm, M is the molecular weight and nr is the number of residues of the protein. However the amino acid residues in GAPDH molecule are varied in the range of 163–179 and also depend on the source of protein. 2.12
Statistical analysis
Statistical analysis was performed using Origin 6 software. Each experiment was performed 3 to 5 times and results are expressed as mean SD and Student’s t-test for significance was performed and p o 0.05 was considered significant. Cellular viability data show representative data of at least three independent experiments.
3. Results and discussion We have prepared a series of water-soluble, monodispersed, highly luminescent, thiol-capped CdX (X = S, Te) quantum dots via a one-pot synthesis route (see the ESI† for details).19,20 The use of 3-mercapto propionic acid (MPA) and N-acetyl-L-cysteine (NAC) as capping agents to bind to the QDs through –SH exposes –COOH and –NH2 groups on the surface of QDs, respectively (Fig. 1a and b). Transmission electron microscopy (TEM) reveals an average particle size of 3 1 nm, which is in agreement with the size determined from the respective absorption onset (see the ESI,† Fig. S1). These QDs offer dimensional matching with proteins revealing a new pathway for controlling
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Fig. 1 Schematic surface structure of CdX QDs synthesized using (a) MPA and (b) NAC as capping agent. (c) Effect of CdSMPA on R-GAPDH (black asterisk and line), N-GAPDH (blue square and line), 3MC-GAPDH (red dots and line) and EAC-GAPDH (magenta stars and line). (d) The L–B double reciprocal plot of R-GAPDH in absence of CdSMPA (black squares and line) and presence of 9.8 nM (red dots and line) and 19 nM (blue stars and line) CdSMPA with various concentrations of GAP. A decrease in Vmax values suggests noncompetitive inhibition mechanism. (e) Dixon plot for R-GAPDH in which measurement of the hydrolysis of the substrate at four different GAP concentrations, 0.04 mM (black squares and line), 0.06 (blue stars and line), 0.1 (green dots and line) and 0.2 mM (red diamonds and line) with CdSMPA concentrations. (f) L–B double reciprocal plot for N-GAPDH in presence of 9.8 nM CdSMPA (red dots and line) and absence of CdSMPA (black squares and line) with various concentrations of GAP.
protein behavior through surface interactions via electrostatic forces and dynamic physicochemical interactions.24 We have studied selective interactions between the functionalized QDs and enzymes taken from normal and malignant origin under physiological conditions. We used GAPDH, a glycolytic enzyme from rabbit skeletal muscle keeping in mind that the structure of rabbit-muscle GAPDH shares 91% sequence identity with the human GAPDH enzyme.25 Since the active site of GAPDH is surrounded by positively charged amino acid residues, the negatively surface-functionalized QDs can effectively block the active site. Subsequently, the accessibility of negatively charged substrates (such as GAP) to the catalytic centre is reduced, resulting in the inhibition of GAPDH activity depending on the concentration of MPA coated QDs in the range of 1–60 nM (enzyme activity monitored through GAPDH catalyzed conversion of NAD+ to NADH at 340 nm, see the ESI,† Fig. S2). Fig. 1c illustrates the effect of MPA coated CdS QDs (CdSMPA) on the activity of GAPDH from rabbit skeletal muscle (R-GAPDH),
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purified GAPDH from normal mice tissues (N-GAPDH), 3MCinduced tumor-bearing mice sarcoma tissue (3-MC GAPDH) and Ehrlich ascites carcinoma (EAC) cell (EAC-GAPDH) (see the ESI,† Fig. S3–S5).26,27 Maximum inactivation (97–100%) of GAPDH activity from the EAC cell was observed with IC50 values of 3.2 nM (3.1–3.5 nM, n = 3) and 6.3 nM (6.0–7.5 nM, n = 3) for 3MC-GAPDH. In contrast, CdSMPA QDs inactivate N-GAPDH and R-GAPDH at higher concentration of QDs (IC50 values of B11 nM for N-GAPDH and 15 nM for R-GAPDH). We have carried out similar tests using NAC (CdSNAC) and cysteine (CdSCys) coated CdS QDs to compare with CdSMPA. CdSMPA showed the strongest inhibition up to B89% on R-GAPDH whereas CdSNAC and CdSCys show 82% and 70% inhibition, respectively (see the ESI,† Fig. S6a). A similar trend is observed for other chalcogenide QDs namely CdTeMPA, CdTeNAC and CdTeCys (see the ESI,† Fig. S6b) suggesting that MPA coated QDs are the most efficient inhibitors owing to the presence of stronger electrostatic interactions between the carboxylate end groups of CdX QDs and cationic residues in the periphery of the active site of GAPDH. The observation is further supported by the zeta potential measurements of respective QDs (see the ESI,† Table S1). In order to probe that inhibition arises due to not only the capping agent end group, but also the effect of QDs, we have carried out control experiments adding individual precursors; Cd++ ions, MPA, NAC and Cys subjected to GAPDH under similar experimental conditions. None of these show any inhibition for GAPDH in the concentration range (see the ESI,† Fig. S7). For CdXMPA, MPA form a highly negatively charged surface by exposing –COOH groups which can interact more strongly with enzyme. Note that this feature is not feasible for pure MPA since thiols have a stronger affinity towards protein molecules, limiting the site selective specificity.24 In order to obtain insight to the inhibition mechanism of GAPDH, we examined the effect of CdSMPA on R-GAPDH, N-GAPDH and 3-MC GAPDH with respect to NAD and GAP substrates. Incubation of R-GAPDH with CdSMPA showed a dose-dependent inhibition which is largely recovered with increasing GAP concentration (Fig. 1d). When the surface charge and structure of inhibitor mimics the substrate molecule, inhibitors may bind to enzymes via non-covalent interactions following competitive or non-competitive inhibition. The Lineweaver–Burk (L–B) plots for R-GAPDH (Fig. 1d) show a non-competitive type of inhibition, suggesting that CdSMPA may not affect directly substrate binding to the active site of the enzyme (see the ESI,† Fig. S8). However, this decreases Vmax (determined from the intercept of the L–B plot) resulting in modification of the enzyme structure.28 This process blocks possibilities of enzyme binding to substrates, reducing enzyme activity and the rate of chemical reaction of the enzyme and substrate. We have calculated the equilibrium constant (Ki) from a Dixon plot29 (Fig. 1e). The Ki value of 21 0.3 mM for R-GAPDH is much lower than the effective concentration of GAP (2.5–25 mM) suggesting a stronger affinity of CdSMPA towards GAPDH in comparison to the substrate. A competitive inhibition with Ki B 39 0.2 mM is observed for N-GAPDH (Fig. 1f). According to the Michaelis–Menten treatment of a reversible competitive inhibitor, the Michaelis constant (Km) may vary
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with QD concentration (see the ESI,† Fig. S9).30 The unaffected Vmax suggests that CdSMPA interferes with substrate binding during the catalytic event, however, it does not bind to the enzyme–substrate complex. In contrast, GAPDH from tumor muscles show irreversible inhibition of enzyme activity by CdSMPA (see the ESI,† Fig. S10). The QDs may react with amino acid side chains of the enzyme molecule to form adducts, specifically altering the active site of enzymes without affecting the protein structure.31 We have tested the inhibition of both normal and malignant GAPDH in presence of CdSMPA at IC50 values with increasing concentration of substrate (GAP). In case of N-GAPDH and R-GAPDH, 50% inhibition of activity was reversibly regained with increasing concentration of GAP (see the ESI,† Fig. S10a). However, for EAC and 3-MC GAPDH activities are not recovered, suggesting a CdSMPA-induced irreversible inhibition (see the ESI,† Fig. S10b). The differential mode of interaction may be attributed due to the presence of different amino acid residues at the active site of the enzyme molecules. Earlier reports suggest that GAPDH from normal tissues is a homotetramer of four identical 35 kDa subunits and each subunit consists of 330 amino acid residues. The cysteine, which remains in the 149 position (Cys-149), is the most reactive among chemically reactive lysine, tyrosine and histidine residues near to the active site (see the ESI,† Fig. S11).32 In case of N-GAPDH and R-GAPDH, Cys-149 is supposed to be the target for QDs, however, for EAC-GAPDH, lysine residues are susceptible to surface-functionalized QDs.26,27 This indicates that the QD binding site of GAPDH from malignant cells is significantly different from that of the enzymes from other normal sources. The GAPDH purified from EAC cells as well as sarcoma mice muscle is a heterodimer of 55 kDa and 33 kDa along with the presence of a critical lysine residue in the active site of the EAC cell enzyme (see the ESI,† Fig. S12). The partial sequences of the 33 kDa and 55 kDa subunits of sarcoma GAPDH are different in comparison to the partial sequence of other four subunit/proteins as reported in the earlier literature (see the ESI,† Table S2). The above observations suggest that CdSMPA inhibits GAPDH activity via differential mechanistic pathways, due to structural and active site modification. We measured the viability of EAC and sarcoma-180 cell lines by a trypan blue exclusion assay. A concentration-dependent reduction of EAC and sarcoma-180 cell viability with IC50 values of 10–15 mg mL1 for CdSMPA was observed (Fig. 2a and b). Interestingly, QDs were found to be lethal for both the EAC and sarcoma cells, but did not show a significant cytotoxic effect for normal cells up to a 50 mg mL1 concentration (Fig. 2c). We have also tested the cytotoxicity of CdSMPA against normal myoblast cells by the standard MTT assay, which relies on the metabolic activity of cells. The activity of the mitochondrial dehydrogenase enzyme of the viable cells and the amount of formazan produced by the reduction of a soluble tetrazolium salt is proportional to the number of cells alive. Reduction of the absorbance value (at 570 nm) of formazone can be attributed to the killing or inhibition of cells by QDs. It is evident from Fig. 2c that the myoblast cells were found to be 490% viable relative to control cells at a concentration up to 50 mg mL1 for CdSMPA.
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Fig. 2 Effect of CdSMPA on (a) EAC and (b) sarcoma-180 cell lines. Cells were treated with CdSMPA at concentrations of 0, 1, 5, 10, 25, 50 and 100 mg mL1 for 1 hour. Quantum dots reduced the cell viability of both EAC and Sarcoma-180 cells with IC50 values of 10–15 mg mL1 CdSMPA. (c) Effect of CdSMPA on normal myoblast (C2C12) mouse cell line. The myoblast cells were incubated with CdSMPA at concentrations of 0, 1, 2, 5, 10, 25, 50 and 100 mg mL1 for 24 hours. QDs have no significant cytotoxic effect for normal cells up to 50 mg mL1 concentration. Data are the mean SD for three independent experiments.
Additionally, we examined the cell morphology in a monolayer culture after treating with different concentrations of CdSMPA (Fig. 3). Microscopic observations showed no distinct morphological changes for normal myoblast cells treated with 50 mg mL1 CdSMPA, indicating normal myoblast cells remain healthy after treating with CdSMPA up to this dose (Fig. 3a–c). In contrast, a dramatic change in the cellular shape of both malignant EAC cells and sarcoma 180 cells are observed after 1 hour treatment with 50 mg mL1 CdSMPA (Fig. 3d–i). The cytotoxic effects of CdSMPA on exposure to normal myoblast mouse cells demonstrated a less significant inhibition of mitochondrial functions at concentrations below 100 mg mL1, whereas less than 10% EAC cells and sarcoma cells were found to be viable after treatment with a 50 mg mL1 concentration of CdSMPA, as determined by a trypan blue exclusion assay (Fig. 2a and b). A similar trend was observed for human cervical cancer cells, where CdSMPA can inhibit cancer cells selectively. These observations suggest that the tumor cells are much more sensitive to functionalized QDs than normal cells. Although GAPDH is expressed in all cells and tissues, malignant cells preferentially contain overexpressed GAPDH. We have observed that QDs show high affinity for malignant GAPDH in comparison to N-GAPDH. When cancer cells are exposed to QDs, loss of cellular GAPDH activity causes metabolic perturbation during glycolysis. This suggests a possible mechanism of impaired energy homeostasis during QDs-mediated cellular injury to the process of cell dysfunction and death, which may find implementation in anticancer therapy.33 Since inhibition of GAPDH leads to the decrease of glycolysis rates, we have measured
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Fig. 3 QDs-induced morphological change of myoblast mouse cells, EAC and Sarcoma-180 cells by phase contrast microscopy. Bright field image of normal myoblast mouse cell (a) without CdSMPA, (b) incubated with 50 mg mL1 of CdSMPA for 12 hours and (c) incubated with 50 mg mL1 of CdSMPA for 24 hours. Bright field image of EAC cell (d) in absence of CdSMPA, (e) presence of CdSMPA (50 mg mL1) for 30 minutes and (f) presence of CdSMPA (50 mg mL1) for 1 hour. Bright field image of Sarcoma-180 cell (g) in absence of CdSMPA, (h) presence of CdSMPA (50 mg mL1) for 30 minutes and (i) presence of CdSMPA (50 mg mL1) for 1 hour. The bright field image shows that, after treatment with CdSMPA, both EAC and Sarcoma180 cells showed swelling (yellow arrows) and irregularities in the plasma membrane and formation of blebs/vacuoles (red arrows). Images were acquired at 10 magnification. Error bar is 2 mm.
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enzyme for energy (ATP) production and blocks the cellular respiration of sarcoma cells (see the ESI,† Table S3). The electron transport chain in the mitochondria generates chemical energy in the form of ATP from adenosine diphosphate and inorganic phosphate via an electrochemical proton gradient and is composed of four membrane-bound complexes such as complex I, complex II, complex III and complex IV. Fig. 4 shows that QDs inhibit a-OG dependent respiration of sarcoma mitochondria. When succinate (5 mM), a substrate for complex II, was added to the system, respiration starts immediately and could be inhibited by malonate (0.5 mM), a known inhibitor of succinate dehydrogenase. These observations suggest that QDs inhibit complex I of the respiratory chain of sarcoma mitochondria because a-OG and pyruvate plus malate donate electrons to complex I, whereas succinate donates electrons to complex II bypassing complex I. The restoration of respiratory activity by succinate indicates that QDs have no effect on the other mitochondrial complexes II, III and IV. Thus, the effect of QDs on GAPDH reveals a link to the differential toxicity observed for normal and malignant cells implying CdSMPA as potential inhibitor for malignant GAPDH. Generally, the process of binding of an inhibitor to an enzyme at the active or other preferential sites induces a conformational change in the enzyme. In order to understand the extent of conformational changes in enzymes after inhibition with QDs, we adopteded circular dichroism (CD) spectroscopy in the UV range (Fig. 5). The CD spectra of the GAPDH show two well defined minima at 208 nm and 222 nm, respectively, which are typical characteristics of proteins containing a-helical secondary structure.34 The secondary structure of GAPDH for N-GAPDH, R-GAPDH, 3MC-GAPDH, and EAC-GAPDH in the presence of
Fig. 4 Oxygen consumption by mice sarcoma tissue mitochondria. Mitochondria were isolated from malignant muscle tissues and the effect of CdSMPA was investigated by polarographic studies. The values along the oxygraph tracing represent nmol oxygen consumption per minute per mg protein. The addition of different compounds is indicated by the arrow. These observations suggest that CdSMPA inhibits complex I of the respiratory chain of sarcoma mitochondria.
the inhibitory effect of CdSMPA on the respiration of intact cells using an oxygen electrode (Fig. 4). Indeed, CdSMPA inhibited respiration of EAC cells and sarcoma cells within a certain time frame (see the ESI,† Table S3). Interestingly, CdSMPA inhibits the mitochondrial complex-I of sarcoma cells, which is a key
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Fig. 5 (a) CD spectra of N-GAPDH in the absence (black squares) and presence of CdSMPA (red circle). (b) CD spectra of R-GAPDH in the absence (black hexagons) and presence of CdSMPA (red circles). (c) CD spectra of 3-MC GAPDH in the absence (black circles) and presence of CdSMPA (red circles). (d) CD spectra of EAC GAPDH in the absence (black stars) and presence of CdSMPA (red circles). The dichroism spectroscopy suggests that the structure of GAPDH remains unaffected during both reversible and irreversible inactivation of enzyme by QDs.
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CdSMPA remains similar after treatment with QDs, implying that the conformations of these GAPDHs remain practically unaltered (Fig. 5). One might not expect a structural change for N-GAPDH or R-GAPDH (Fig. 5a and b) considering the reversible inactivation by QDs. However, a structural change is expected for both 3MC-GAPDH and EAC-GAPDH since the QDs induced irreversible inactivation (Fig. 5c and d). The protein conformation of 3MC-GAPDH shows little change, possibly due to the reversible inactivation of enzyme activity by QDs. The a-helical content of GAPDH protein after treated with QDs were calculated in terms of mean residue ellipticity (MRE) values at 208 nm.37,38 The helicity of GAPDH does not significantly change (81% remaining) which suggests preservation of the protein structure after treatment with QDs. The dichroism spectroscopy univocally suggests that the structure of GAPDH remains the same for both reversible and irreversible inactivation by the QDs. This observation is further corroborated by bright field (BF) imaging of GAPDH in the presence of QDs (see the ESI,† Fig. S13). The BF images of GAPDH do not show any structural change in the presence of QDs suggesting that the inactivation occurs before noticeable conformational change of the enzyme as a whole.35,36 Though the specific activity of GAPDH decreases drastically with increasing QD concentration, the overall preservation of the enzyme secondary structure suggests that diffusion limitation is the main reason for enzyme inhibition. Owing to the presence of electrostatic interactions, further particle clustering and steric hindrance, QDs immobilize in the active site of the enzyme limiting free diffusion of the substrates into the active site.
4. Conclusions In summary, we have shown a novel enzyme inhibition route to modulate enzyme activities using different functionalized metal chalcogenide QDs. The QDs inhibit reversibly GAPDH enzyme activity from normal sources, whereas malignant cells and sarcoma tissues are affected in an irreversible way. The malignant GAPDH are irreversibly inhibited at a significantly low concentration of QDs, an observation that may contribute to their unique therapeutic benefits, including rapid onset of inhibition with greater potency. Such an irreversible inhibition mechanism may also find usefulness in drug discovery owing to the specificity to the target enzyme between the host and pathogens. Since GAPDH is present in all normal tissues, our route of controlled inhibition of enzyme activities towards malignant GAPDH is generic and will find broad application. Specifically, the inhibition of cancer cells GAPDH by QDs through controlled inhibition of mitochondrial function may lead to a deleterious effect on cancer cells. Such a selective inhibition and targeting of GAPDH based on direct chemical interaction in malignancy is unique.
Acknowledgements Financial support under a grant from the DST, India is gratefully acknowledged. S. G., A. H. K. and M. R. gratefully acknowledge
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the CSIR, India for financial support. The authors thank A. Ghosh for providing cellular respiration measurements.
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Modulation of glyceraldehyde-3-phosphate dehydrogenase activity by surface functionalized quantum dots† Srabanti Ghosh,*a Manju Ray,b Mahua Rani Das,c Adrita Chakrabarti,a Ali Hossain Khan,a D. D. Sarmad and Somobrata Acharya*a Enzymatic regulation is a fast and reliable diagnosis tool via identification and design of inhibitors for modulation of enzyme function. Previous reports on quantum dots (QDs)–enzyme interactions reveal a protein-surface recognition ability leading to promising applications in protein stabilization, protein delivery, bio-sensing and detection. However, the direct use of QDs to control enzyme inhibition has never been revealed to date. Here we show that a series of biocompatible surface-functionalized metal–chalcogenide QDs can be used as potent inhibitors for malignant cells through the modulation of enzyme activity, while normal cells remain unaffected. The in vitro activity of glyceraldehyde-3phosphate dehydrogenase (GAPDH), an enzyme involved critically in the glycolysis of cancer cells, is
Received 16th August 2013, Accepted 10th December 2013
inactivated selectively in a controlled way by the QDs at a significantly low concentration (nM).
DOI: 10.1039/c3cp53489h
mechanisms owing to the site-specific interactions, enabling control over the inhibition kinetics. These complementary loss-of-function probes may offer a novel route for rapid clinical diagnosis of malignant
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cells and biomedical applications.
Cumulative kinetic studies delineate that the QDs undergo both reversible and irreversible inhibition
1. Introduction The emergence of the design of enzyme modulators provides a potent tool to target malignant cells via blocking an enzyme activity that influences metabolic imbalance.1 The Warburg effect, which explains the increased glycolytic capacity of cancer cells, is the most fundamental metabolic alteration during malignant transformation.2 The increased glycolytic capacity of a cancer cell is associated with mitochondrial defects and the abnormal expression of metabolic enzymes.3 Glyceraldehyde-3phosphate dehydrogenase (GAPDH) is one of the key enzymes in the glycolytic cascade, which constitute a novel target for anticancer therapy due to overexpression in many cancer tissues such as lung cancer, prostatic adenocarcinoma, renal cell carcinoma, breast carcinoma cells, hepatocellular carcinoma etc. without affecting their normal counterparts.4–6 Normal cells adapt a
Centre for Advanced Materials, Indian Association for the Cultivation of Science, Kolkata 700032, India. E-mail: [email protected], [email protected] b Division of Molecular Medicine, Bose Institute, Kolkata 700 054, India c Biological Chemistry, Indian Association for the Cultivation of Science, Kolkata-700032, India d Solid State and Structural Chemistry Unit & Centre for Condensed Matter Theory Indian Institute of Science, Bangalore 560012, India † Electronic supplementary information (ESI) available: Additional discussions and TEM images. See DOI: 10.1039/c3cp53489h
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alternative metabolic pathways to sustain their energy requirements during increased glycolytic activity during which ATP generation through mitochondrial oxidative phosphorylation is compromised. Consequently, frequent mitochondrial DNA mutations, malfunction of the electron transport chains, aberrant enzyme expression in energy metabolism and insufficient oxygen availability in the cellular microenvironment are generally evidenced in a variety of human cancers.7 The inner core tumor cells naturally become hypersensitive to materials as a result of abnormal cellular bioenergetics, which block the glycolytic pathways at different steps. Hence, the development of novel glycolytic inhibitors can be utilized as a new class of cancer biomarkers and in a variety of therapeutic applications.8 GAPDH inhibitors can block the conversion of the oxidative phosphorylation of glyceraldehyde 3-phosphate (GAP) to 1,3-bisphosphoglycerate in the sixth step of glycolysis, which is effective in the inhibition of cellular respiration causing cancer cell death.9,10 Thus, the glycolytic pathway provides a biochemical basis for novel therapeutic strategies to prevent cancer cell growth by the pharmacological inhibition of glycolysis. Several molecules exhibit promising anticancer activities as single units or in combination with other therapeutic modalities based on enzyme inhibition to influence different cellular processes such as apoptosis and angiogenesis.11,12 Unfortunately, these molecular inhibitors generally do not show
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specificity for the inhibition of enzyme activity.13 Thus, an alternative strategy to achieve a target-specific agent with efficient therapeutic selectivity that differentiates between normal and cancer cells in course of biochemical metabolism is crucial. Several studies have suggested that the surface modified quantum dots (QDs) exhibit improved binding with enzyme molecules which may allow a better understanding of the underlying kinetic mechanisms at the nanoparticle interface for designing QDs based biological tools.14,15 Recently, the use of QDs together with bio-molecules as diagnostic tools has shown major advantages over traditional chemotherapeutic compounds in cancer research.16,17 However, the use of QD often leads to aggregation upon conjugation with protein, limiting the effectiveness of disease diagnosis.18 Here we report on the use of metal chalcogenide QDs as potential enzyme inhibitor with differential enzymatic regulation. In vitro inhibition studies of GAPDH in presence of QDs suggest that the binding of QDs to the enzyme molecules slows down the rate of the enzyme catalyzed reaction probing QDs as a potential enzyme inhibitor. We investigated the mechanistic pathways for enzyme inhibition using GAPDH from normal and tumour associated mammalian tissues, respectively. In vitro enzyme kinetic analyses show that QDs have a marked affinity towards enzyme molecules in comparison to the substrates following a differential modulation of enzyme activities via reversible or irreversible targeted inhibition. Spectroscopic evidence reveals that the binding of QDs with GAPDH retains the protein structure both for normal and tumour associated tissues. Our results highlight new insights into the underlying correlation between the antiproliferative activity and inhibition of enzyme activity by QDs in cancer cells without alteration of the structural conformation.
2. Experimental 2.1
Reagents
3-Mercaptopropionic acid, L-cysteine hydrochloride, cadmium chloride hexahydrate, glycine, magnesium chloride, sodium hydroxide, ethylenediaminetetraacetic acid (EDTA), p-nitrophenyl phosphate, sodium chloride (NaCl), potassium chloride (KCl) and disodium hydrogen phosphate (Na2HPO4) were purchased from Merck, Germany. N-Acetyl-L-cysteine (NAC), tellurium powder, sodium borohydrate (NaBH4), triethylamine hydrochloride, glyceraldehyde-3-phosphate (GAP), nicotinamide adenine dinucleotide (NAD), b-mercaptoethanol, glycerol, glyceraldehyde-3phosphate dehydrogenase (GAPDH) from rabbit skeletal muscle, 3-methylcholanthrene, a-oxoglutarate, ADP (disodium salt), succinate, malonate, and pyridoxal 5 0 -phosphate (PP) were obtained from Sigma Chemical Co., St. Louis, MO, USA. Sephadex G-50 was purchased from Calbiochem and Pharmacia Fine Chemicals, Sweden. All the chemicals were of analytical grade or highest purity available and were used as-obtained. Milli-Q water (Millipore) was used as a solvent. 2.2
Synthesis of thiol-capped quantum dots
CdS and CdTe QDs were synthesized following the method as reported earlier with trivial modification.19,20 In a typical synthesis,
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an aqueous solution of Cd2+ ion (2 102 M) is dissolved in 100 mL water and 5 102 M of the thiol stabilizer, such as 3-mercapto propionic acid (MPA), N-acetyl-L-cysteine (NAC), cysteine hydrochloride (Cys) used as capping agents followed by adjusting pH 11 with 1 M NaOH solution. Then a freshly prepared aqueous solution of H2S of known concentration (estimated iodometrically using sodium arsenite) was added in a stoichiometric amount to the N2 purged Cd–thiolate complex solution. For CdTe QDs, a freshly prepared aqueous solution of sodium hydrogen telluride (NaHTe) was added to maintain the final molar ratio of Cd : thiols : Te – 1 : 2 : 0.5. The resultant solution was refluxed at 100 1C and aliquots were collected at various time intervals having different sizes of QDs. To remove the excess Cd–thiolate complexes at the end of the synthesis, 2-propanol was added to the reaction mixture to precipitate CdTe and CdS QDs. The as-prepared product was freeze-dried under vacuum for further experiments. 2.3
GAPDH assay
GAPDH activity was measured spectrophotometrically by monitoring the reduction of NAD+ to NADH at 340 nm. The reaction was carried out at 25 1C and was initiated by the addition of 1–5 mg of the enzyme to the reaction mixture containing 50 mmol triethanolamine buffers, 50 mmol Na2HPO4, 0.2 mmol EDTA, 1 mmol NAD and 0.5 mmol of GAP. To monitor the reaction, the increase in absorbance at 340 nm due to the formation of NADH from NAD was noted at 30 second intervals, the values remaining almost linear for 5 minutes. One unit of activity is defined as the amount of enzyme required to convert 1 mmol of NAD+ to NADH per minute at standard assay conditions (pH 8.5, 25 1C). 2.4
Development of sarcoma in mice
Animal experiments were carried out in accordance with the guidelines of the institutional ethics committee (IEC). Appropriate precautions were taken to minimize pain or discomfort to animals. 2.5
Development of tumour
Sarcoma tissue was developed in the left hind leg of mice by intramuscular injection of 3-methyl-cholanthrene. The carcinogen was dissolved in olive oil by placing it in warm water-bath and 0.1 mL of olive oil containing 0.2 mg of the carcinogen was injected into each mouse thrice with a one-week interval. After 12–14 weeks a full-grown tumour was developed. The malignancy was confirmed by histological examination where differentiated muscle cells are conspicuously visible in normal mouse muscle, and highly differentiated sarcoma tissue was observed where tumour was developed by 3-methyl-cholanthrene. 2.6 Purification of GAPDH from normal muscle, sarcoma tissue and Ehrlich ascites carcinoma (EAC) cells 15 gm of either normal mice muscle or sarcoma tissue was homogenized in an Omni GLH International homogenizer with four volumes of 50 mM triethanolamine-HCl buffer, pH 7.4 containing 10 mM EDTA and 10 mM b-mercaptoethanol. The tissue and the buffer were pre-cooled and all operations were carried out at 0–4 1C. From the homogenate, GAPDH was
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purified by gel filtration and ion exchange chromatography. The enzyme fractions after the DEAE-sephacel column step were purified 28 and 96 fold for normal muscle and sarcoma tissues, respectively. The specific activities of GAPDH at this stage were 112 for normal muscle and 313 for sarcoma tissue.
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2.7
Cell lines
Sarcoma 180 cells and EAC cells were maintained in the peritoneal cavity of Swiss albino female mice by weekly inoculation of 2 106 cells. 2.8
Cytotoxicity assays (MTT assay)
A murine cell line was used to determine the cytotoxicity of QDs. Cell toxicity was determined using the MTT assay, based on the reduction of a soluble tetrazolium salt by mitochondrial dehydrogenase of the viable cells to a water-insoluble coloured product (i.e. formazan).21 The activity of the enzyme and the amount of formazan produced is proportional to the number of cells alive. Briefly, C2C12 myoblast mice cells were seeded at a density of 2 105 cells per well in a 96-well microtiter plate for 18–24 h before the assay. The cells were incubated for 24 h at 37 1C under 5% CO2 in the presence of a varying concentration of quantum dots. Then, 50 ml of a 5 mg ml1 MTT (Sigma; St Louis, MO) was added to each well, followed by 4 h of incubation at 37 1C. The formazan crystals were then solubilized in 200 ml DMSO. The optical density (OD) at 570 nm was measured using an automated BioTeks Elisa Reader. The number of surviving cells were expressed as percentage viability = (the absorbance of the treated cells) background/the absorbance of the control (untreated cells) background 100. The cell toxicity of the extracted contents from scaffolds was rated as follows: severe (o30%); moderate (30–60%); slight (60–90%); or non-cytotoxic (490%) of MTT activity, compared to the control cells cultured in an extract-free medium. 2.9
Trypan blue exclusion assay
To measure the viable cell percentage, trypan blue dye was employed to stain the cells which did not have an intact membrane. The viable cells exclude the dyes and are not stained, which is a standard method to detect cell death. First, EAC and Sarcoma 180 cells were washed with PBS buffer pH 7.4 and then the cells were incubated for 1 h at 37 1C in the presence of varying concentrations of quantum dots. Finally, 10 mL of the trypan blue dye was added to each cellular suspension. The viable cell was counted through a hemocytometer, and the viability values were derived by comparing the samples with the negative control. The morphological changes of the C2C12 myoblast mice cells and malignant cells treated with QDs were visualized with a BX51WI fluorescence microscope (Olympus) equipped with 460–490 nm excitation filter setting and a DP71 digital camera and DP-BSW software for image acquisition. 2.10 Preparation of mitochondria and measurement of mitochondrial respiration Sarcoma tissue was collected and washed in a buffer containing 250 mM sucrose, 1 mM EDTA, 10 mM Tris/HCl pH 7.4 and
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0.1% BSA. After finely mincing the tissue, it was homogenized in 6 vol. of the buffer with 10 up and down strokes of a Potter– Elvehjem homogenizer and centrifuged at 1500g for 5 min. The supernatant was collected and centrifuged at 8000g for 15 min. The pellet was suspended and washed twice with same buffer by centrifuging at 8000g for 15 min and finally suspended in a minimum volume of the buffer. Additionally, mitochondria from EAC cells were prepared essentially by digitonin permeabilization by the method of Moreadith and Fiskum with some minor modifications.22 Mitochondrial oxygen consumption was measured with a Hansatech oxygraph fitted with a Clark electrode.23 The respiratory medium in a total volume of 2 mL contained 125 mM KCl, 10 mM Tris/MOPS (pH 7.4), 1 mM KH2PO4, 1 mM MgCl2, 1 mM EGTA and the respiratory substrates, which were usually 10 mM a-oxoglutarate or 10 mM pyruvate plus 10 mM malonate or 5 mM succinate. The mitochondrial protein in the medium was 0.3–0.6 mg. 2.11
Structural analysis
Circular dichroism (CD) spectra have been recorded by JASCO, CD Spectrometer; model J-815-150S using a 0.1 cm path length quartz cell in a wavelength range between 190 and 250 nm. The results were expressed as molar ellipticity [y] in units of m degree g cm2 dmol1. Molar ellipticity values were obtained using the relation: y = (Myobs)/10clnr where yobs is the observed ellipticity in degrees at a given wavelength, c is the protein concentration in mole per c.c. and l is the length of the light path in cm, M is the molecular weight and nr is the number of residues of the protein. However the amino acid residues in GAPDH molecule are varied in the range of 163–179 and also depend on the source of protein. 2.12
Statistical analysis
Statistical analysis was performed using Origin 6 software. Each experiment was performed 3 to 5 times and results are expressed as mean SD and Student’s t-test for significance was performed and p o 0.05 was considered significant. Cellular viability data show representative data of at least three independent experiments.
3. Results and discussion We have prepared a series of water-soluble, monodispersed, highly luminescent, thiol-capped CdX (X = S, Te) quantum dots via a one-pot synthesis route (see the ESI† for details).19,20 The use of 3-mercapto propionic acid (MPA) and N-acetyl-L-cysteine (NAC) as capping agents to bind to the QDs through –SH exposes –COOH and –NH2 groups on the surface of QDs, respectively (Fig. 1a and b). Transmission electron microscopy (TEM) reveals an average particle size of 3 1 nm, which is in agreement with the size determined from the respective absorption onset (see the ESI,† Fig. S1). These QDs offer dimensional matching with proteins revealing a new pathway for controlling
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Fig. 1 Schematic surface structure of CdX QDs synthesized using (a) MPA and (b) NAC as capping agent. (c) Effect of CdSMPA on R-GAPDH (black asterisk and line), N-GAPDH (blue square and line), 3MC-GAPDH (red dots and line) and EAC-GAPDH (magenta stars and line). (d) The L–B double reciprocal plot of R-GAPDH in absence of CdSMPA (black squares and line) and presence of 9.8 nM (red dots and line) and 19 nM (blue stars and line) CdSMPA with various concentrations of GAP. A decrease in Vmax values suggests noncompetitive inhibition mechanism. (e) Dixon plot for R-GAPDH in which measurement of the hydrolysis of the substrate at four different GAP concentrations, 0.04 mM (black squares and line), 0.06 (blue stars and line), 0.1 (green dots and line) and 0.2 mM (red diamonds and line) with CdSMPA concentrations. (f) L–B double reciprocal plot for N-GAPDH in presence of 9.8 nM CdSMPA (red dots and line) and absence of CdSMPA (black squares and line) with various concentrations of GAP.
protein behavior through surface interactions via electrostatic forces and dynamic physicochemical interactions.24 We have studied selective interactions between the functionalized QDs and enzymes taken from normal and malignant origin under physiological conditions. We used GAPDH, a glycolytic enzyme from rabbit skeletal muscle keeping in mind that the structure of rabbit-muscle GAPDH shares 91% sequence identity with the human GAPDH enzyme.25 Since the active site of GAPDH is surrounded by positively charged amino acid residues, the negatively surface-functionalized QDs can effectively block the active site. Subsequently, the accessibility of negatively charged substrates (such as GAP) to the catalytic centre is reduced, resulting in the inhibition of GAPDH activity depending on the concentration of MPA coated QDs in the range of 1–60 nM (enzyme activity monitored through GAPDH catalyzed conversion of NAD+ to NADH at 340 nm, see the ESI,† Fig. S2). Fig. 1c illustrates the effect of MPA coated CdS QDs (CdSMPA) on the activity of GAPDH from rabbit skeletal muscle (R-GAPDH),
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purified GAPDH from normal mice tissues (N-GAPDH), 3MCinduced tumor-bearing mice sarcoma tissue (3-MC GAPDH) and Ehrlich ascites carcinoma (EAC) cell (EAC-GAPDH) (see the ESI,† Fig. S3–S5).26,27 Maximum inactivation (97–100%) of GAPDH activity from the EAC cell was observed with IC50 values of 3.2 nM (3.1–3.5 nM, n = 3) and 6.3 nM (6.0–7.5 nM, n = 3) for 3MC-GAPDH. In contrast, CdSMPA QDs inactivate N-GAPDH and R-GAPDH at higher concentration of QDs (IC50 values of B11 nM for N-GAPDH and 15 nM for R-GAPDH). We have carried out similar tests using NAC (CdSNAC) and cysteine (CdSCys) coated CdS QDs to compare with CdSMPA. CdSMPA showed the strongest inhibition up to B89% on R-GAPDH whereas CdSNAC and CdSCys show 82% and 70% inhibition, respectively (see the ESI,† Fig. S6a). A similar trend is observed for other chalcogenide QDs namely CdTeMPA, CdTeNAC and CdTeCys (see the ESI,† Fig. S6b) suggesting that MPA coated QDs are the most efficient inhibitors owing to the presence of stronger electrostatic interactions between the carboxylate end groups of CdX QDs and cationic residues in the periphery of the active site of GAPDH. The observation is further supported by the zeta potential measurements of respective QDs (see the ESI,† Table S1). In order to probe that inhibition arises due to not only the capping agent end group, but also the effect of QDs, we have carried out control experiments adding individual precursors; Cd++ ions, MPA, NAC and Cys subjected to GAPDH under similar experimental conditions. None of these show any inhibition for GAPDH in the concentration range (see the ESI,† Fig. S7). For CdXMPA, MPA form a highly negatively charged surface by exposing –COOH groups which can interact more strongly with enzyme. Note that this feature is not feasible for pure MPA since thiols have a stronger affinity towards protein molecules, limiting the site selective specificity.24 In order to obtain insight to the inhibition mechanism of GAPDH, we examined the effect of CdSMPA on R-GAPDH, N-GAPDH and 3-MC GAPDH with respect to NAD and GAP substrates. Incubation of R-GAPDH with CdSMPA showed a dose-dependent inhibition which is largely recovered with increasing GAP concentration (Fig. 1d). When the surface charge and structure of inhibitor mimics the substrate molecule, inhibitors may bind to enzymes via non-covalent interactions following competitive or non-competitive inhibition. The Lineweaver–Burk (L–B) plots for R-GAPDH (Fig. 1d) show a non-competitive type of inhibition, suggesting that CdSMPA may not affect directly substrate binding to the active site of the enzyme (see the ESI,† Fig. S8). However, this decreases Vmax (determined from the intercept of the L–B plot) resulting in modification of the enzyme structure.28 This process blocks possibilities of enzyme binding to substrates, reducing enzyme activity and the rate of chemical reaction of the enzyme and substrate. We have calculated the equilibrium constant (Ki) from a Dixon plot29 (Fig. 1e). The Ki value of 21 0.3 mM for R-GAPDH is much lower than the effective concentration of GAP (2.5–25 mM) suggesting a stronger affinity of CdSMPA towards GAPDH in comparison to the substrate. A competitive inhibition with Ki B 39 0.2 mM is observed for N-GAPDH (Fig. 1f). According to the Michaelis–Menten treatment of a reversible competitive inhibitor, the Michaelis constant (Km) may vary
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with QD concentration (see the ESI,† Fig. S9).30 The unaffected Vmax suggests that CdSMPA interferes with substrate binding during the catalytic event, however, it does not bind to the enzyme–substrate complex. In contrast, GAPDH from tumor muscles show irreversible inhibition of enzyme activity by CdSMPA (see the ESI,† Fig. S10). The QDs may react with amino acid side chains of the enzyme molecule to form adducts, specifically altering the active site of enzymes without affecting the protein structure.31 We have tested the inhibition of both normal and malignant GAPDH in presence of CdSMPA at IC50 values with increasing concentration of substrate (GAP). In case of N-GAPDH and R-GAPDH, 50% inhibition of activity was reversibly regained with increasing concentration of GAP (see the ESI,† Fig. S10a). However, for EAC and 3-MC GAPDH activities are not recovered, suggesting a CdSMPA-induced irreversible inhibition (see the ESI,† Fig. S10b). The differential mode of interaction may be attributed due to the presence of different amino acid residues at the active site of the enzyme molecules. Earlier reports suggest that GAPDH from normal tissues is a homotetramer of four identical 35 kDa subunits and each subunit consists of 330 amino acid residues. The cysteine, which remains in the 149 position (Cys-149), is the most reactive among chemically reactive lysine, tyrosine and histidine residues near to the active site (see the ESI,† Fig. S11).32 In case of N-GAPDH and R-GAPDH, Cys-149 is supposed to be the target for QDs, however, for EAC-GAPDH, lysine residues are susceptible to surface-functionalized QDs.26,27 This indicates that the QD binding site of GAPDH from malignant cells is significantly different from that of the enzymes from other normal sources. The GAPDH purified from EAC cells as well as sarcoma mice muscle is a heterodimer of 55 kDa and 33 kDa along with the presence of a critical lysine residue in the active site of the EAC cell enzyme (see the ESI,† Fig. S12). The partial sequences of the 33 kDa and 55 kDa subunits of sarcoma GAPDH are different in comparison to the partial sequence of other four subunit/proteins as reported in the earlier literature (see the ESI,† Table S2). The above observations suggest that CdSMPA inhibits GAPDH activity via differential mechanistic pathways, due to structural and active site modification. We measured the viability of EAC and sarcoma-180 cell lines by a trypan blue exclusion assay. A concentration-dependent reduction of EAC and sarcoma-180 cell viability with IC50 values of 10–15 mg mL1 for CdSMPA was observed (Fig. 2a and b). Interestingly, QDs were found to be lethal for both the EAC and sarcoma cells, but did not show a significant cytotoxic effect for normal cells up to a 50 mg mL1 concentration (Fig. 2c). We have also tested the cytotoxicity of CdSMPA against normal myoblast cells by the standard MTT assay, which relies on the metabolic activity of cells. The activity of the mitochondrial dehydrogenase enzyme of the viable cells and the amount of formazan produced by the reduction of a soluble tetrazolium salt is proportional to the number of cells alive. Reduction of the absorbance value (at 570 nm) of formazone can be attributed to the killing or inhibition of cells by QDs. It is evident from Fig. 2c that the myoblast cells were found to be 490% viable relative to control cells at a concentration up to 50 mg mL1 for CdSMPA.
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Fig. 2 Effect of CdSMPA on (a) EAC and (b) sarcoma-180 cell lines. Cells were treated with CdSMPA at concentrations of 0, 1, 5, 10, 25, 50 and 100 mg mL1 for 1 hour. Quantum dots reduced the cell viability of both EAC and Sarcoma-180 cells with IC50 values of 10–15 mg mL1 CdSMPA. (c) Effect of CdSMPA on normal myoblast (C2C12) mouse cell line. The myoblast cells were incubated with CdSMPA at concentrations of 0, 1, 2, 5, 10, 25, 50 and 100 mg mL1 for 24 hours. QDs have no significant cytotoxic effect for normal cells up to 50 mg mL1 concentration. Data are the mean SD for three independent experiments.
Additionally, we examined the cell morphology in a monolayer culture after treating with different concentrations of CdSMPA (Fig. 3). Microscopic observations showed no distinct morphological changes for normal myoblast cells treated with 50 mg mL1 CdSMPA, indicating normal myoblast cells remain healthy after treating with CdSMPA up to this dose (Fig. 3a–c). In contrast, a dramatic change in the cellular shape of both malignant EAC cells and sarcoma 180 cells are observed after 1 hour treatment with 50 mg mL1 CdSMPA (Fig. 3d–i). The cytotoxic effects of CdSMPA on exposure to normal myoblast mouse cells demonstrated a less significant inhibition of mitochondrial functions at concentrations below 100 mg mL1, whereas less than 10% EAC cells and sarcoma cells were found to be viable after treatment with a 50 mg mL1 concentration of CdSMPA, as determined by a trypan blue exclusion assay (Fig. 2a and b). A similar trend was observed for human cervical cancer cells, where CdSMPA can inhibit cancer cells selectively. These observations suggest that the tumor cells are much more sensitive to functionalized QDs than normal cells. Although GAPDH is expressed in all cells and tissues, malignant cells preferentially contain overexpressed GAPDH. We have observed that QDs show high affinity for malignant GAPDH in comparison to N-GAPDH. When cancer cells are exposed to QDs, loss of cellular GAPDH activity causes metabolic perturbation during glycolysis. This suggests a possible mechanism of impaired energy homeostasis during QDs-mediated cellular injury to the process of cell dysfunction and death, which may find implementation in anticancer therapy.33 Since inhibition of GAPDH leads to the decrease of glycolysis rates, we have measured
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Fig. 3 QDs-induced morphological change of myoblast mouse cells, EAC and Sarcoma-180 cells by phase contrast microscopy. Bright field image of normal myoblast mouse cell (a) without CdSMPA, (b) incubated with 50 mg mL1 of CdSMPA for 12 hours and (c) incubated with 50 mg mL1 of CdSMPA for 24 hours. Bright field image of EAC cell (d) in absence of CdSMPA, (e) presence of CdSMPA (50 mg mL1) for 30 minutes and (f) presence of CdSMPA (50 mg mL1) for 1 hour. Bright field image of Sarcoma-180 cell (g) in absence of CdSMPA, (h) presence of CdSMPA (50 mg mL1) for 30 minutes and (i) presence of CdSMPA (50 mg mL1) for 1 hour. The bright field image shows that, after treatment with CdSMPA, both EAC and Sarcoma180 cells showed swelling (yellow arrows) and irregularities in the plasma membrane and formation of blebs/vacuoles (red arrows). Images were acquired at 10 magnification. Error bar is 2 mm.
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enzyme for energy (ATP) production and blocks the cellular respiration of sarcoma cells (see the ESI,† Table S3). The electron transport chain in the mitochondria generates chemical energy in the form of ATP from adenosine diphosphate and inorganic phosphate via an electrochemical proton gradient and is composed of four membrane-bound complexes such as complex I, complex II, complex III and complex IV. Fig. 4 shows that QDs inhibit a-OG dependent respiration of sarcoma mitochondria. When succinate (5 mM), a substrate for complex II, was added to the system, respiration starts immediately and could be inhibited by malonate (0.5 mM), a known inhibitor of succinate dehydrogenase. These observations suggest that QDs inhibit complex I of the respiratory chain of sarcoma mitochondria because a-OG and pyruvate plus malate donate electrons to complex I, whereas succinate donates electrons to complex II bypassing complex I. The restoration of respiratory activity by succinate indicates that QDs have no effect on the other mitochondrial complexes II, III and IV. Thus, the effect of QDs on GAPDH reveals a link to the differential toxicity observed for normal and malignant cells implying CdSMPA as potential inhibitor for malignant GAPDH. Generally, the process of binding of an inhibitor to an enzyme at the active or other preferential sites induces a conformational change in the enzyme. In order to understand the extent of conformational changes in enzymes after inhibition with QDs, we adopteded circular dichroism (CD) spectroscopy in the UV range (Fig. 5). The CD spectra of the GAPDH show two well defined minima at 208 nm and 222 nm, respectively, which are typical characteristics of proteins containing a-helical secondary structure.34 The secondary structure of GAPDH for N-GAPDH, R-GAPDH, 3MC-GAPDH, and EAC-GAPDH in the presence of
Fig. 4 Oxygen consumption by mice sarcoma tissue mitochondria. Mitochondria were isolated from malignant muscle tissues and the effect of CdSMPA was investigated by polarographic studies. The values along the oxygraph tracing represent nmol oxygen consumption per minute per mg protein. The addition of different compounds is indicated by the arrow. These observations suggest that CdSMPA inhibits complex I of the respiratory chain of sarcoma mitochondria.
the inhibitory effect of CdSMPA on the respiration of intact cells using an oxygen electrode (Fig. 4). Indeed, CdSMPA inhibited respiration of EAC cells and sarcoma cells within a certain time frame (see the ESI,† Table S3). Interestingly, CdSMPA inhibits the mitochondrial complex-I of sarcoma cells, which is a key
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Fig. 5 (a) CD spectra of N-GAPDH in the absence (black squares) and presence of CdSMPA (red circle). (b) CD spectra of R-GAPDH in the absence (black hexagons) and presence of CdSMPA (red circles). (c) CD spectra of 3-MC GAPDH in the absence (black circles) and presence of CdSMPA (red circles). (d) CD spectra of EAC GAPDH in the absence (black stars) and presence of CdSMPA (red circles). The dichroism spectroscopy suggests that the structure of GAPDH remains unaffected during both reversible and irreversible inactivation of enzyme by QDs.
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CdSMPA remains similar after treatment with QDs, implying that the conformations of these GAPDHs remain practically unaltered (Fig. 5). One might not expect a structural change for N-GAPDH or R-GAPDH (Fig. 5a and b) considering the reversible inactivation by QDs. However, a structural change is expected for both 3MC-GAPDH and EAC-GAPDH since the QDs induced irreversible inactivation (Fig. 5c and d). The protein conformation of 3MC-GAPDH shows little change, possibly due to the reversible inactivation of enzyme activity by QDs. The a-helical content of GAPDH protein after treated with QDs were calculated in terms of mean residue ellipticity (MRE) values at 208 nm.37,38 The helicity of GAPDH does not significantly change (81% remaining) which suggests preservation of the protein structure after treatment with QDs. The dichroism spectroscopy univocally suggests that the structure of GAPDH remains the same for both reversible and irreversible inactivation by the QDs. This observation is further corroborated by bright field (BF) imaging of GAPDH in the presence of QDs (see the ESI,† Fig. S13). The BF images of GAPDH do not show any structural change in the presence of QDs suggesting that the inactivation occurs before noticeable conformational change of the enzyme as a whole.35,36 Though the specific activity of GAPDH decreases drastically with increasing QD concentration, the overall preservation of the enzyme secondary structure suggests that diffusion limitation is the main reason for enzyme inhibition. Owing to the presence of electrostatic interactions, further particle clustering and steric hindrance, QDs immobilize in the active site of the enzyme limiting free diffusion of the substrates into the active site.
4. Conclusions In summary, we have shown a novel enzyme inhibition route to modulate enzyme activities using different functionalized metal chalcogenide QDs. The QDs inhibit reversibly GAPDH enzyme activity from normal sources, whereas malignant cells and sarcoma tissues are affected in an irreversible way. The malignant GAPDH are irreversibly inhibited at a significantly low concentration of QDs, an observation that may contribute to their unique therapeutic benefits, including rapid onset of inhibition with greater potency. Such an irreversible inhibition mechanism may also find usefulness in drug discovery owing to the specificity to the target enzyme between the host and pathogens. Since GAPDH is present in all normal tissues, our route of controlled inhibition of enzyme activities towards malignant GAPDH is generic and will find broad application. Specifically, the inhibition of cancer cells GAPDH by QDs through controlled inhibition of mitochondrial function may lead to a deleterious effect on cancer cells. Such a selective inhibition and targeting of GAPDH based on direct chemical interaction in malignancy is unique.
Acknowledgements Financial support under a grant from the DST, India is gratefully acknowledged. S. G., A. H. K. and M. R. gratefully acknowledge
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the CSIR, India for financial support. The authors thank A. Ghosh for providing cellular respiration measurements.
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