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endoplasmic reticulum Ca2+-ATPase and results in cell Ca2+ imbalance
Archives of Biochemistry and Biophysics 570 (2015) 58–65
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Chelerythrine inhibits the sarco/endoplasmic reticulum Ca2+-ATPase and results in cell Ca2+ imbalance Saulo Martins Vieira a,c,⇑, Vanessa Honorato de Oliveira a, Raphael do Carmo Valente a,b, Otacílio da Cruz Moreira d, Carlos Frederico Leite Fontes a,⇑, Julio Alberto Mignaco a,⇑ a
Instituto de Bioquímica Médica Leopoldo de Meis, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Instituto de Biofísica Carlos Chagas Filho, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Laboratório de Toxinologia, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil d Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil b c
a r t i c l e
i n f o
Article history: Received 29 October 2014 and in revised form 9 February 2015 Available online 23 February 2015 Keywords: SERCA ATPase Chelerythrine PBMC Cytotoxicity Calcium
a b s t r a c t The isoquinoline alkaloid chelerythrine is described as an inhibitor of SERCA. The ATPase inhibition presented two non-competitive components, Ki1 = 1, 2 lM and Ki2 = 26 lM. Conversely, chelerythrine presented a dual effect on the p-nitrophenylphosphatase (pNPPase) of SERCA. Ca2+-dependent pNPPase was activated up to 5 lM chelerythrine with inhibition thereafter. Ca2+-independent pNPPase was solely inhibited. The phosphorylation of SERCA with ATP reached half-inhibition with 10 lM chelerythrine and did not parallel the decrease of ATPase activity. In contrast, chelerythrine up to 50 lM increased the phosphorylation by Pi. Cross-linking of SERCA with glutaraldehyde was counteracted by high concentrations of chelerythrine. The controlled tryptic digestion of SERCA shows that the low-affinity binding of chelerythrine evoked an E2-like pattern. Our data indicate a non-competitive inhibition of ATP hydrolysis that favors buildup of the E2-conformers of the enzyme. Chelerythrine as low as 0.5–1.5 lM resulted in an increase of intracellular Ca2+ on cultured PBMC cells. The inhibition of SERCA and the loss of cell Ca2+ homeostasis could in part be responsible for some described cytotoxic effects of the alkaloid. Thus, the choice of chelerythrine as a PKC-inhibitor should consider its potential cytotoxicity due to the alkaloid’s effects on SERCA. Ó 2015 Elsevier Inc. All rights reserved.
Introduction SERCA1 (sarco/endoplasmic reticulum Ca2+-ATPase), (E.C. 3.6.3.8.) is a P-type ATPase directly involved in cell Ca2+ responses and homeostasis. This enzyme translocates two Ca2+ ions from the cytoplasm into the endoplasmic reticulum using the energy derived from the hydrolysis of ATP, thereby contributing to decrease the amount of calcium in the cytoplasm. Thus, cell responses dependent on high
⇑ Corresponding authors at: Laboratório de Estrutura e Regulação de Proteínas e ATPases, Instituto de Bioquímica Médica Leopoldo de Meis, UFRJ, CCS, sala H2-026, Avenida Brigadeiro Trompowsky s/n° Ilha do Fundão, Rio de Janeiro, RJ 21941-590, Brazil. E-mail addresses: [email protected] (S.M. Vieira), [email protected]. br (C.F.L. Fontes), [email protected] (J.A. Mignaco). 1 Abbreviations used: SERCA, sarco/endoplasmic reticulum Ca2+-ATPase; pNPPase, pnitrophenylphosphatase; pNPP, p-nitrophenylphosphate; CHE, chelerythrine; DTT, DLdithiotreitol; PMSF, phenylmethylsulfonyl fluoride; MTT, 1-(4,5-dimethylthiazol-2yl)-3,5-diphenylformazan; SRV, Sarcoplasmic reticulum vesicles; PBMC, Peripheral Blood Mononuclear Cells; FBS, fetal bovine serum; PTP, permeability transition pore; PMCA, plasma membrane Ca2+-ATPase. http://dx.doi.org/10.1016/j.abb.2015.02.019 0003-9861/Ó 2015 Elsevier Inc. All rights reserved.
cytoplasmic calcium concentrations, like muscle contraction, are terminated. As for other P-type ATPases, SERCA’s catalytic cycle presents two main conformational states, E1 and E2, and the enzyme becomes reversibly phosphorylated at an aspartate (D351) within the DKTGTLT sequence common to all P-ATPases [1–4]. The phosphorylated enzyme (E1–P) occludes two calcium ions coordinated by polar and ionic side chains of aminoacid residues present at the transmembrane helices M4, M5, M6 and M8 [5] and after conversion to the E2–P form the calcium sites, now facing the luminal side of the reticulum, lose affinity and release the ion inside the ER. The enzyme is then dephosphorylated regenerating the free enzyme ready for a new cycle (Scheme 1) [6,7]. This dephosphorylation is the rate-limiting step of the cycle [8]. The dephosphorylation is controlled by the conserved TGES motif, which coordinate and activate one H2O molecule for nucleophilic attack by abstracting a proton [9]. It has been demonstrated that protonation of the residues at the empty Ca2+ binding sites in E2–P is mandatory for normal dephosphorylation of E2–P, and that mutations at the Ca2+ binding sites could affect this protonation [7,10]. Besides ATP, SERCA can hydrolyze pseudosubstrates, like p-nitrophenylphosphate (pNPP) [11,12] or
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preparation performed as [28] was chosen due to purity reasons. Protein concentrations were determined according to Lowry et al. [29] using bovine serum albumin as standard. pNPPase activity
Scheme 1. The simplified E1/E2 cycle of SERCA (Adapted from 6,7).
acetylphosphate [12,13], and their hydrolysis is also able to sustain Ca2+ transport [13,14]. SERCA, as other members of the P-type ATPase family, shows a biphasic kinetic behavior for the hydrolysis of ATP [15,16]. At micromolar ATP concentrations an increase of hydrolytic activity is observed and the phosphoenzyme builds up. The increase of ATP to millimolar concentrations leads to higher rates of ATP hydrolysis yet without a further increase on the E–P amount. This kinetic behavior for long time raised a debate about whether one or two ATP-binding sites existed for each SERCA monomer. Structural evidence supports the one-site hypothesis, and thus the second ATPase burst must result from a kinetic effect prompted by ATPbinding to the already phosphorylated catalytic portion [17,18]. Chelerythrine (CHE) is an isoquinoline alkaloid commonly found in plants from the families Papaveraceae, Fumariaceae and Rutaceae [19]. Chelerythrine and similar alkaloids are used in the Traditional Chinese Medicine and, since some years ago, in medicinal chemistry because of their ample biological activities. Chelerythrine is a well characterized, cell permeable and potent inhibitor of PKC [20]. As chelerythrine effects on isolated PKC isoforms are well established, many reports describe that, for several cell lineages, chelerythrine (and related alkaloids) elicit cell responses attributed mainly to the inhibition of PKC [20–24]. However, authors also report other effects of chelerythrine, such as DNA damage, antifungal, antibacterial and antitumor properties, which have no direct relation to the inhibition of PKC [20]. Indeed, several authors claim that many of the alkaloid’s effects could be due to the interaction of this compound with numerous cell proteins, therefore pointing for the need of better control and interpretation of experiments when using whole or leaky cells [25,26]. The main goal of the present study was to show that besides its well known effects on PKC, chelerythrine is also a powerful inhibitor of the SERCA activity in the same concentration range widely used for the inhibition of PKC. Therefore, SERCA inhibition by chelerythrine could be a primary cause of cytotoxicity in target cells by the resulting loss of the cell calcium homeostasis. Materials and methods Reagents EDTA, DL-dithiotreitol (DTT), EGTA, HEPES, ATP (sodium salt), phosphatidylcholine, phenylmethylsulfonyl fluoride (PMSF), C12E8, 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), A23187, Fura-2AM and TRIZMA-base were purchased from SIGMA (St. Louis, MO, USA). Chelerythrine chloride was from either SIGMA or Calbiochem with similar results. [32P]Pi was from the Instituto de Pesquisas Energéticas e Nucleares – IPEN (São Paulo, SP, Brazil). Enzyme preparation Sarcoplasmic reticulum vesicles (SRV) were prepared from rabbit hind leg skeletal muscle as described by [27] and stored in liquid nitrogen. In the case of proteolysis experiments, Meissner
p-Nitrophenylphosphatase (pNPPase) activity was measured at 37 °C in media containing 20 mM Tris–Cl (pH 7.5), 0.5 mM EGTA or 0.05 mM CaCl2, 120 mM KCl, 10 mM MgCl2, 20 lg mL 1 SRV protein, 10 lM A23187, variable chelerythrine concentrations, and 3 mM pNPP. After 20–40 min, the reactions (0.5 mL) were quenched with 0.1 mL of 0.3 N NaOH, and 0.4 mL of deionized water, p-nitrophenol was measured by its absorption at 425 nm, assuming an extinction coefficient of 1.1 104 M 1 cm 1. Effect of chelerythrine chloride on the Ca2+-ATPase activity The Ca2+-ATPase activity was assayed at 37 °C in media with 100 mM Tris–Cl (pH 7.5), 4 mM MgCl2, 80 mM KCl, 0.05 mM CaCl2 and 28 lg mL 1 SRV protein, and different concentrations of chelerythrine. The reaction was initiated by addition of 3 mM Na+-ATP and stopped after 3 min by addition of 100 lL of 1% SDS. Activity was determined by counting the amount of radioactive Pi released from [c-32P]ATP. [c-32P]ATP was synthesized accordingly to the method of [30], with modifications described in [31]. The contamination of the [c-32P]ATP with free 32Pi never exceeded the limit of 0.5–1.0%. Phosphoenzyme determinations SERCA was phosphorylated by ATP in media containing 20 mM Tris–Cl (pH 7.5), 120 mM KCl, 10 mM MgCl2, 0.05 mM CaCl2, 100 lg mL 1 SRV protein, and the chelerythrine concentrations indicated. The reaction was started with 10 lM [c-32P]ATP and quenched after 5 s with 1 volume of cold 0.4 mM PCA plus 1 mM Pi. For the phosphorylation by [32P]Pi, 100 lg mL 1 SRV protein in an E2-favoring reaction buffer (20 mM MES-pH 6.0-with 10 mM MgCl2, 2 mM EGTA, and the indicated chelerythrine concentrations) was incubated and 4 mM [32P]Pi were added. Reactions were quenched as above. Samples were filtered in 0.45 lm Millipore filters HAWP246 employing a Hoeffer vacuum system apparatus and washed 3 times with the quenching solution. The radioactivity remaining in the filter (specifically bound phosphoenzyme) was counted using a Packard Tri-carb 2600 Liquid Scintillation Counter. Digestion of SERCA by trypsin in presence of chelerythrine chloride proteolysis was done in 50 mM Tris–Cl (pH 7.5), and 2 mg mL 1 SERCA was preincubated for 5 min with 1, 10, 50 and 100 lM chelerythrine chloride or with either 5 mM ADP or 5 mM sodium orthovanadate (final volume of 100 lL). Digestion was performed with 12 lg mL 1 trypsin for 2 min at 37 °C. The reaction was stopped with 10 lL of Laemmli denaturing buffer (6, final 62.5 mM Tris–Cl pH 6.8, 2% SDS, 5% b-mercaptoethanol) and stored in ice. SDS–PAGE was performed accordingly [32], on 10–21% acrylamide continuous gradient gels. 80 lg of total protein were applied to each well and slab gels run for 4 h at 120 V and 80 mA. The gels were stained with Coomassie blue. Intramolecular cross-linking with glutaraldehyde Reaction buffer contained 30 mM MOPS/Tris (pH 8.5, 7.5 or 6.5), 0.2 M sucrose, 80 mM KCl, 0.05 mM CaCl2, chelerythrine from 0.5 to 100 lM. After pre-incubation with chelerythrine for 5 min, 0.2 mg mL 1 SRV were incubated with 0.3 mM glutaraldehyde for 90 min at room temperature as described by [33]. The cross-
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linking reaction was quenched with 1/6 volume of denaturing buffer (6, final 62.5 mM Tris–Cl pH 6.8, 2% SDS and 5% b-mercaptoethanol). SDS–PAGE was performed on 7.5% gel acrylamide. The gel was stained with Coomassie blue and scanned. Band densities were calculated with the ImageJ software. Collection of blood samples and separation of monocytes Twenty milliliters of blood samples were obtained from healthy volunteers using sodium heparin (Roche, Brazil) as anticoagulant. Peripheral Blood Mononuclear Cells (PBMC) were separated by density gradient through centrifugation for 30 min at 400 g using 100% Ficoll–Histopaque reagent (GE, USA). After separation, cells were washed three times with PBS buffer for 8 min at 220 g. Intracellular calcium measurements 1 106 PBMC cells/mL were incubated with 1.5 lM FURA-2 AM in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) for 40 min at room temperature in the dark. Then cells were washed twice with PBS and suspended in PBS with 1 mM CaCl2. The [Ca2+]i was quantified according to [34] using an FP-6300 spectrofluorometer (JASCO, Japan) set to kex = 340/362 nm, kem = 540 nm, Slitex = 10 nm and Slitem = 5 nm, with constant stirring at 37 °C. After 2 min a concentrated volume of chelerythrine was added and the fluorescence shift recorded. Results Incubation of SR vesicles with increasing amounts of chelerythrine resulted in a marked, progressive inhibition of the Ca2+ATPase activity when ATP was the substrate. This culminated in a maximal inhibition of the ATPase activity in presence of 200 lM chelerythrine, with an IC501.5 lM (Fig. 1). A small fraction of the total ATPase activity could not be inhibited. This residual activity was probably due to the existence of a Ca2+-insensitive, Mg2+-ATPase activity typical of the sarcoplasmic reticulum [35] that is resilient to classical P-type ATPase inhibitors, and seems also not to be affected by chelerythrine. The inhibition of SERCA was partially (ca. 30%) reversed by centrifugation and washing, and the addition of thiol reducers did not contribute to any further reversal, evidencing a strong but not covalent binding to the protein (data not shown).
Fig. 1. Inhibition of SERCA by chelerythrine. SRV were incubated in presence of the alkaloid, and the ATPase activity measured in presence of increasing concentrations of chelerythrine. The assay media contained 0.028 mg/mL of SRV protein and 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 150 and 200 lM of chelerythrine. The nominal control activity of the Ca2+-ATPase was 3.2 lmoles/mg/min and the plot was presented as relative activities in comparison with the activity of the control samples. Each condition represents the mean ± SE (n = 4).
Fig. 1 clearly points to the existence of two components with distinct affinities for the inhibition of SERCA ATPase activity by chelerythrine. Therefore, a Dixon plot was made in order to identify each component and their relative affinities. This result is shown in Fig. 2, where the two components were clearly distinguished and we could thus calculate that the affinity for binding differs by one order of magnitude, with Ki1 = 1.2 lM e Ki2 = 26 lM. The profile of the Dixon plot already suggested a non-competitive type of inhibition for both high and low affinity inhibitory components. This non-competitive character was also confirmed by the substrate dependence for ATPase activity in absence or presence of chelerythrine (Fig. 3). The kinetic parameters shown in Table 1 make evident that although the Vmax decreased for any region of the ATP dependence curve there is a negligible decrease of the affinity for ATP at the catalytic site and a sizeable but not important decrease of the affinity at the ‘‘regulatory’’ site. SERCA is known to support high rates of pNPP hydrolysis in presence of Ca2+, developing a cycle much alike the ATPase one and also supporting Ca2+ transport. On the other hand, in absence of Ca2+ the enzyme is also able to promote a non-productive, futile hydrolysis of pNPP solely by the E2 conformer [14]. Chelerythrine clearly inhibited the unproductive pNPP hydrolysis conducted by the E2 conformer in absence of Ca2+ (Fig. 4). Although also seeming to present two components, this inhibition impaired only ca. 50% of the initial activity with as much as 50 lM chelerythrine. Nevertheless, in presence of Ca2+ the alkaloid promoted an opposite, dual effect on the dose–response curve for chelerythrine of the pNPPase activity, activating the phosphatase with a peak at 5–10 lM chelerythrine and progressively decreasing the activity thereafter, although with a drastically shifted IC50 if compared to the values found for the ATPase activity (Fig. 4). In order to figure out where chelerythrine would bind to SERCA, and whether this binding induced any conformational selection, we used the chemical modification approach of intramolecular cross-linking between residues at the nucleotide site with glutaraldehyde described by [33]. This cross-linking alters the mobility of SERCA in SDS–PAGE from 110 kDa to a band with apparent M.R. 125 kDa. Chelerythrine within 0.5–10 lM, the range of the high affinity component for inhibition of the SERCA ATPase activity, did not substantially impair formation of the 125 kDa band at any pH tested (6.5/7.5/8.5). However, a small decrease of crosslinking which saturated around 5–10 lM chelerythrine, became evident when the pH shifted from 6.5 to 8.5. Distinctive protection against the cross-linking occurred only at pH 7.5 and 8.5 and once the concentration of chelerythrine increased towards the low affinity component for inhibition of the ATPase activity (Fig. 5A–D). It is noteworthy that this protection at higher chelerythrine was not evident at pH 6.5 (Fig. 5A and D), which may suggest a dependence either on the conformation of the enzyme or on the ionization state of chelerythrine, or both, for the low affinity binding of the alkaloid. As Ca2+-ATPase and Ca2+-pNPPase activities in principle follow a similar cycle, we measured the phosphoenzyme levels for the steady-state in presence of chelerythrine using ATP as substrate. With 10 lM ATP as substrate no significant changes on the phosphoenzyme level were observed until beyond 5 lM chelerythrine (Fig. 6) when the ATPase activity was inhibited over 60% (see Figs. 1 and 3), suggesting that the nucleotide binding and phosphoryl transfer steps were not much affected. However, with higher chelerythrine the phosphoenzyme was significantly impaired. This suggests that binding of a second chelerythrine molecule to SERCA might impede ATP binding and phosphoryl transfer to the enzyme by shifting the enzyme to the E2-form, incompetent to be phosphorylated by 10 lM ATP [36]. The stabilization of the E2-related conformer might also be provoked by the binding of a second molecule of chelerythrine with lower affinity (which seems
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Fig. 2. Dixon plot of the inhibition of SERCA by chelerythrine. SRV were incubated with increasing concentrations of the alkaloid: (A) 0.2; 0.5; 1; 2 and 5 lM; (B) 0.5, 10, 20, 50; 100; 150 and 200 lM both performed with two concentrations of substrate (1 or 3 mM Na+-ATP). The assay media contained 0.028 mg/mL of SRV in buffer 100 mM Tris– HCl pH 7.5; 80 mM KCl; 4 mM MgCl2, 0.05 mM CaCl2, at 37 °C. Representative Dixon plots were obtained by the means of three independent chelerythrine inhibition experiments, error bars mean SE.
Fig. 3. ATP-dependence of SERCA with chelerythrine. The ATPase activity was measured in absence (-d-) or presence of 1 lM chelerythrine (-s-). The assay media contained 0.020 mg/mL SRV protein and increasing concentrations of ATP ranging from 1 lM to 2 mM. The kinetic parameters were calculated with the best fit of a double-michaelian curve using the kinetic module of Sigmaplot 8.0. Results are mean ± SE (n = 3).
Table 1 Kinetic parameters for inhibition of SERCA by 1 lM chelerythrine.
Control Chelerythrine
Km1 (lM)
Vmax1 (nmol/ mg/min)
Km2 (lM)
Vmax2 (nmol/ mg/min)
0.49 ± 0.19 0.58 ± 0.34
143.3 ± 32.5 62.2 ± 8.0
397.4 ± 62.6 762.6 ± 57.0
1707.6 ± 72.3 1283.7 ± 33.8
to be corroborated by data obtained in Figs. 1, 2, 4 and 5), in which an inhibitory effect on phosphatase activity and in E–P accumulation is observed with chelerythrine concentrations from 10 to 50 lM. The proteolysis of SERCA by trypsin is a method capable of detecting changes in the conformation of the sarco/endoplasmic reticulum Ca2+-ATPase as a result of incubation with a series of ligands and conditions. Trypsin first cleaves SERCA at a highly susceptible site at Arg505 of the N-domain (T1) producing two major fragments A and B. Further cleavage of fragment A at Arg198 of the A-domain (T2) yields subfragments A1 and A2 [37,38]. The rate at which these fragments are generated and further degraded by
Fig. 4. Effects of chelerythrine on the pNPPase activity of SERCA. The pNPPase activity of SRV was measured in presence of increasing concentrations of the alkaloid, either in presence of 50 lM CaCl2 (-d-) or 1 mM EGTA (-s-). Results are mean ± SE (n = 3).
trypsin depends on the conformational state of SERCA. We therefore performed proteolysis in the presence of diverse ligands known to shift SERCA conformation [33,39] toward the E1 (ADP) or the E2 (orthovanadate or 2 mM Ca2+) state and compared the digestion pattern obtained as a result of increasing amounts of chelerythrine. The results obtained are highly suggestive that, in low concentrations (up to 10 lM), chelerythrine binding to SERCA does not contribute to shift the enzyme conformation to anything different from the E1 pattern; but increasing the alkaloid concentration from 10 to 100 lM changes the scenario and ends up by giving an E2-like proteolysis pattern (Fig. 7). Control experiments (not shown) confirmed that the proteolytic activity of the trypsin was not changed by the presence of orthovanadate, ADP or chelerythrine ranging from 1 to 100 lM. Hitherto kinetic and structural data indicated that the first chelerythrine binding component although not directly shifting the conformational state had serious implications in impairing ATP hydrolysis. Conversely, based on the glutaraldehyde cross-linking and on the proteolysis assays the low affinity binding component appeared to shift enzyme conformation to a possible E2-like state. Therefore we verified the consequence of chelerythrine on the backdoor phosphorylation of SERCA by inorganic phosphate, which
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Fig. 5. Cross-link formation in SERCA by glutaraldehyde. 0.2 mg/mL of SRV were mixed with the indicated concentrations of chelerythrine and after that samples were incubated with 0.3 mM glutaraldehyde for 90 min. The results at different pH’s are presented as (A) pH = 6.5, (B) pH = 7.5, (C) pH = 8.5, and (D) density of bands. The gel staining was Coomassie blue-R (panels A–C). In panel (D), the symbols are (-d-) pH = 8.5, (-s-) pH = 7.5 and (-.-) pH = 6.5. The relative density of band was calculated and normalized to the 125 kD band in the absence of chelerythrine, which was indexed as 100%.
Fig. 6. Inhibition of SERCA ATP-driven phosphorylation by chelerythrine. SRV were incubated in presence of the alkaloid, and the phosphoenzyme formed quantified. The assay media contained 0.028 mg/mL of SRV protein, 10 lM [c-32P]ATP (specific activity of 2 105 cpm/nmol) and 1, 2, 5, 10, 20 and 50 lM of chelerythrine. The reactions were started by addition of the substrate, and after 2 s quenched with 1 vol. of perchloric acid 0.4 M (final 0.2 M). Each condition represents the mean ± S.D. (n = 3).
is possible when the enzyme is incubated at slightly acidic pH and in absence of Ca2+. In this situation the enzyme is phosphorylated by Pi on the E2 conformation [17,40]. Not unexpectedly, we found that when phosphorylation was performed with Pi as substrate (from the E2 state), the phosphoenzyme formation was increased by chelerythrine at any concentration of the alkaloid, indicating an effect of the alkaloid, shifting the enzyme to an E2-like
Fig. 7. Proteolysis of SERCA in presence of chelerythrine. SERCA (1.0 mg/mL) was incubated with chelerythrine, ADP or sodium orthovanadate, and digested by trypsin (0.012 mg/mL) for 2 min at 37 °C at pH 7.5. Proteolysis was stopped by the addition of electrophoresis sample buffer and equal amounts (40 lg) of digested protein was applied at a gradient (10–21%) SDS–PAGE. Lanes: 1 – SERCA (Meissner) control, 2 – Meissner + trypsin, 3–6 – same as 2 with increasing concentrations of chelerythrine (1/10/50/100), 7 – 5 mM ADP and 8 – 5 mM orthovanadate.
conformation that facilitates phosphorylation by Pi, increasing the observed E–P levels (Fig. 8). Although the direct binding and the inhibition of SERCA by chelerythrine were clear, we still had no evidence that this inhibition
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Scheme 2. pH-dependent (acid–base) equilibrium between the charged (iminium ion) and the uncharged (pseudobase) form of chelerythrine. The reported pKR+ values of chelerythrine in aqueous solutions range from 7.5 to 9.2 (Vlcˇková et al., 2004).
Fig. 8. Accumulation of Pi-driven phosphoenzyme in the presence of chelerythrine. The phosphorylation by [32P]Pi (specific activity of 2 105 cpm/nmol) was performed with 100 lg/mL SRV protein in an E2-favoring reaction buffer (20 mM MES pH 6.0, with 10 mM MgCl2 and the indicated chelerythrine concentrations). The enzyme was incubated at 37 °C and the reaction was started by the addition of 4 mM [32P]Pi. Reactions were stopped after 1 min by the addition of 1 vol. of 0.4 M perchloric acid. Results are mean ± S.D. (n = 3).
would occur in whole cells and contribute to impair cell Ca2+ homeostasis. We then labeled quiescent fresh PBMC cells with the cytosolic Ca2+ sensor Fura2-AM and incubated them with chelerythrine between 0.5 and 1.5 lM, which are usually employed in PKC inhibition assays (reported IC50 for PKC is 0.6 lM). It was remarkable that almost immediately the [Ca2+]i started to increase steadily for several minutes after the addition of the low chelerythrine amounts (Fig. 9). In the time lapse acquired the [Ca2+]i was increased by P60% using chelerythrine concentrations that are elsewhere reported to cause cytotoxicity [41–43].
Discussion Chelerythrine is an isoquinoline alkaloid widely used as inhibitor of PKC and related calcium-mediated signal transduction in cells. The reported EC50 value for PKC inhibition by chelerythrine is around 0.6 lM, and its effects seem to be due to its binding to the catalytic domain of PKC promoting a non-competitive inhibition with respect to ATP, but with a competitive inhibition regarding to phosphate acceptors like histone H3s [20]. From the chemical point of view, chelerythrine can interconvert between a quaternary iminium form (cationic form) and
pseudobase/hydroxide adducts (neutral form), with a pKa ranging 7.5–9.2 (Scheme 2) [44–46]. At physiological pH apparently chelerythrine permeates the cell membrane in the form of a nonpolar pseudobase [47,48]. Slaninová et al. [48] reported that chelerythrine enters the cell in the form of yellow-orange vesicles, indicating that the quaternary adduct form of the alkaloid enters the cell by pinocytosis. Once inside the cell, chelerythrine distributes within cell compartments [48,49]. The iminium bond of the quaternary form (C6@N+) is susceptible to nucleophilic attack, and this reaction is essential to the effect of the reported inhibition of SH-proteins [50]. In our experiments, performed at pH 7.5, we did not find any evidence pointing to the reaction of thiol groups of SERCA with chelerythrine. Isoquinoline alkaloids have been used in a wide range of applications, ranging from toothpaste additives as bacteriostatic agents [50] to potentially antitumor drugs [41,51–53]. Several reports describing pro-apoptotic effects of these alkaloids were published, and most of them implicate mitochondrial function alterations as the main causative factor of apoptosis [42,54,55]. A possible direct effect of chelerythrine in membrane ion-transporting ATPases has, though, been underestimated in the literature, with some older reports of sanguinarine and other alkaloids inhibiting Na,KATPase [56,57]. A recent paper describes the inhibitory effect of sanguinarine and its dihydroderivative on the Na,K-ATPase [58], and these authors claimed that the E1-form has the sanguinarine binding site in a more accessible state than the E2-form of the enzyme. We show in this work that chelerythrine, in concentrations very near to those used to inhibit PKC, strikingly impaired SERCA’s activity. SERCA inhibition could lead to a deregulated Ca2+ homeostasis in cells treated with the alkaloid and could at least in part be responsible for some of the reported effects of chelerythrine in cell cultures [42,54,59]. The mechanism by which chelerythrine inhibits SERCA is still not completely defined. SERCA is well characterized as presenting
Fig. 9. Intracellular increase of calcium in presence of chelerythrine. After incubation of PBMC with FURA-2 AM, cells were washed and resuspended with PBS and 1 mM CaCl2. Arrow indicates addition of chelerythrine after 2 min of fluorescence analysis in several chelerythrine conditions 0, 0.5, 1 and 1.5 lM. The asterisk represents the [Ca2+]i after exposition to 50 nM thapsigargin during 30 min as a control. In the case of absence of CHE, an equivalent amount of water was added during fluorescence measurements. (B) [Ca2+]i at 30 min, comparison between chelerythrine and thapsigargin. The results are representative of five separate experiments.
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two Km’s for ATP [15,16]. Therefore, it was of interest to gather whether the inhibition of the ATPase activity was competitive with the binding of ATP, since the inhibition of PKC by chelerythrine was already described to be non-competitive [20]. The ATP-dependence and kinetic parameters for the Ca2+-ATPase activity inhibited by chelerythrine showed that Km1 and Km2 were not significantly shifted, and the Dixon plot confirmed the non-competitive character of SERCA’s ATPase activity. Thus, our data support that low concentrations of chelerythrine inhibit SERCA and decrease the overall hydrolysis rate by a high affinity, non-competitive binding mechanism. Similarly, pNPP hydrolysis by the E2 conformer of the enzyme is also impaired at low chelerythrine concentrations, suggesting that phosphorylation/dephosphorylation steps in the E2 form might be slower. A conflicting result is obtained for pNPP hydrolysis in presence of Ca2+, where a slight, but consistent activation is seen with the same low chelerythrine concentrations. On the other hand, at low chelerythrine the SERCA phosphoenzyme levels formed with ATP were not altered, evidencing that, for instance, ATP binds and phosphorylates the enzyme, but further reaction steps are impaired. And as well, phosphorylation by Pi is enhanced with a biphasic profile. Therefore, the profile of all the activity curves and phosphorylation results altogether support that SERCA apparently presents two binding sites for the alkaloid. However, probably the kinetic effects observed depend on the rate-limiting step and on the enzyme conformers involved in the hydrolysis cycle for the different substrates and, for instance, also on the conformational changes involved when Ca2+ is transported. The consequences of chelerythrine binding to SERCA on the intramolecular cross-linking and on the digestion pattern by trypsin are suggestive about chelerythrine-induced conformational changes of the enzyme. As shown in Figs. 5 and 7, low chelerythrine does not seem to majorly prevent cross-linking or to modify the trypsin digestion pattern. Moreover, concentrations of the alkaloid above 10 lM result in gradual impairment of cross-linking and also result in an E2-like digestion pattern. Therefore, at least for chelerythrine concentrations corresponding to the 2nd, low affinity binding component that inhibit the ATP activity (apart from other kinetic effects), it is possible to suggest that chelerythrine induced an enzyme conformational drift to the E2 state. This proposal is also supported by the effects of high concentrations of chelerythrine inhibiting the ATP-driven phosphorylation and promoting Pi-driven phosphoenzyme accumulation (Figs. 6 and 8). The involvement of Ca2+ signaling in cell death is well recognized. Persistent high intracellular Ca2+ leads to mitochondrial Ca2+ overload, which in turn leads to opening of the mitochondrial permeability transition pore (PTP), dissipation of electrochemical gradients, mitochondrial swelling, and release of cytochrome c and other apoptotic signals, and ultimately leads to cell death [60,61]. The induction of apoptosis by inhibition of SERCA with thapsigargin, leading to Ca2+ stress, is documented [61] and results in an outcome comparable to the described for chelerythrine or the related alkaloid sanguinarine. Independent of the mechanism involved in the inhibition of SERCA, there is a close proximity between the concentrations of chelerythrine sufficient to impair the enzyme catalytic function and those which cause the rapid and continued increase in PBMC cytosolic calcium (see Fig. 9). Therefore, our results are suggestive of the direct involvement of SERCA inhibition by chelerythrine in triggering the increase in cell calcium that will, for instance, lead to apoptosis and cell death, which was reported employing diverse cell models [41,42,59]. Our observations do not rule out that besides SERCA other transporters involved in cell Ca2+ homeostasis could be (directly or indirectly) affected by chelerythrine. For instance, the plasma membrane Ca2+-ATPase (PMCA), a member of the P-type ATPase family that pumps Ca2+ outward the cell [62], presents a high
degree of structural and functional homology with both SERCA and Na, K-ATPase, and could therefore be a potential target of the alkaloid. Preliminary results show that the PMCA-driven ATP hydrolysis is in fact impaired by chelerythrine, and this is a subject of current investigation in our laboratory. In addition, the outward Ca2+ pumping capacity of the Na+/Ca2+ exchanger [62] could be affected by a slight, but consistent inhibition of the Na,K-ATPase by chelerythrine (roughly 20% inhibition with 2 lM of the alkaloid, not shown) that could also change the Na+ balance in the long term. No matter the fractional contribution of each mentioned mechanism, the outcome shall be the same: increased cell calcium triggering cell death. Our work raises the concern that, besides the inhibition of PKC by chelerythrine, that certainly will be of serious consequence to cells, a direct and relevant inhibition of SERCA will result in an uncontrolled cell calcium increase thereby triggering mitochondrial events that will ultimately lead to cell death by apoptosis in either healthy or disease-model cell types. Since the high affinity Ki observed for the chelerythrine-dependent inhibition of ATP hydrolysis by SERCA was 1.2 lM, which is only two times greater than the reported Ic50 of 0.66 lM for PKC inhibition, the safety for the use of chelerythrine as a specific pharmacological inhibitor of PKC is at least questionable. We suggest that more careful attention should be taken when testing chelerythrine and related alkaloids in mammalian cells, since chelerythrine is widely marketed and employed as a specific PKC inhibitor during signal-transduction based studies. Acknowledgments The helpful technical assistance of M.Sc. Mônica M. Freire and Geiza L. Fraga is greatly acknowledged. We also thank the formerly undergraduate students Ana Paula Carvalho and Catarina C. P. Prima for their contribution in the beginning of this project. A.P.C. and C.C.P.P. were recipients of undergraduate fellowships from CNPq. V.H.O. and O.C.M. were recipients of graduate fellowships from CNPq. C.F.L.F. is recipient of a research fellowship from CNPq. This work was supported by grants from CNPq, CAPES and FAPERJ. References [1] K.B. Axelsen, M.G. Palmgren, J. Mol. Evol. 46 (1998) 84–101. [2] J.V. Moller, B. Juul, M. le Maire, Biochim. Biophys. Acta 1286 (1996) 1–51. [3] J.P. Andersen, B. Vilsen, H. Nielsen, J.V. Møller, Biochemistry 25 (1986) 6439– 6447. [4] E. Carafoli, Annu. Rev. Physiol. 53 (1991) 531–547. [5] C. Toyoshima, M. Nakasako, H. Nomura, H. Ogawa, Nature 405 (2000) 647–655. [6] L. de Meis, A.L. Vianna, Annu. Rev. Biochem. 48 (1979) 275–292. [7] A.G. Lee, J.G. East, Biochem. J. 356 (2001) 665–683. [8] D. Lewis, R. Pilankatta, G. Inesi, G. Bartolommei, M.R. Moncelli, F. TadiniBuoninsegni, J. Biol. Chem. 287 (2012) 32717–32727. [9] C. Olesen, T.L. Sorensen, R.C. Nielsen, J.V. Moller, P. Nissen, Science 306 (2004) 2251–2255. [10] J.P. Andersen, Biosci. Rep. 15 (1995) 243–261. [11] J.M. Ribeiro, E.S. Aragão, A.L. Vianna, An. Acad. Bras. Cienc. 52 (1980) 403–409. [12] M. Ushimaru, Y. Fukushima, Biochem. Biophys. Res. Co. 353 (2007) 799–804. [13] L. Meis, W. Hasselbach, J. Biol. Chem. 246 (1971) 4759–4763. [14] J.A. Mignaco, O.H. Lupi, F.T. Santos, H. Barrabin, H.M. Scofano, Biochemistry 35 (1996) 3886–3891. [15] S. Verjovski-Almeida, G. Inesi, Biochim. Biophys. Acta 558 (1979) 119–125. [16] J.S. Taylor, D. Hattan, J. Biol. Chem. 254 (1979) 4402–4407. [17] P. Champeil, S. Riollet, S. Orlowski, F. Guillain, C.J. Seebregts, D.B. McIntosh, J. Biol. Chem. 263 (1988) 12288–12294. [18] J.M. Autry, J.M. Rubin, B. Svensson, J. Li, D.D. Thomas, J. Biol. Chem. 287 (2012) 39070–39082. [19] M. Wink (Ed.), Alkaloids. Biochemistry Ecology and Medical Applications, Plenum Press, London, 1998. pp. 265–298. [20] J.M. Herbert, J.M. Augereau, J. Gleye, J.P. Maffrand, Biochem. Biophys. Res. Commun. 172 (1990) 993–999. [21] S.J. Chmura, M.E. Dolan, A. Cha, H.J. Mauceri, D.W. Kufe, R.R. Weichselbaum, Clin. Cancer Res. 6 (2000) 737–742. [22] A. Shrivastava, Indian J. Exp. Biol. 45 (2007) 755–763.
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Chelerythrine inhibits the sarco/endoplasmic reticulum Ca2+-ATPase and results in cell Ca2+ imbalance Saulo Martins Vieira a,c,⇑, Vanessa Honorato de Oliveira a, Raphael do Carmo Valente a,b, Otacílio da Cruz Moreira d, Carlos Frederico Leite Fontes a,⇑, Julio Alberto Mignaco a,⇑ a
Instituto de Bioquímica Médica Leopoldo de Meis, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Instituto de Biofísica Carlos Chagas Filho, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Laboratório de Toxinologia, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil d Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil b c
a r t i c l e
i n f o
Article history: Received 29 October 2014 and in revised form 9 February 2015 Available online 23 February 2015 Keywords: SERCA ATPase Chelerythrine PBMC Cytotoxicity Calcium
a b s t r a c t The isoquinoline alkaloid chelerythrine is described as an inhibitor of SERCA. The ATPase inhibition presented two non-competitive components, Ki1 = 1, 2 lM and Ki2 = 26 lM. Conversely, chelerythrine presented a dual effect on the p-nitrophenylphosphatase (pNPPase) of SERCA. Ca2+-dependent pNPPase was activated up to 5 lM chelerythrine with inhibition thereafter. Ca2+-independent pNPPase was solely inhibited. The phosphorylation of SERCA with ATP reached half-inhibition with 10 lM chelerythrine and did not parallel the decrease of ATPase activity. In contrast, chelerythrine up to 50 lM increased the phosphorylation by Pi. Cross-linking of SERCA with glutaraldehyde was counteracted by high concentrations of chelerythrine. The controlled tryptic digestion of SERCA shows that the low-affinity binding of chelerythrine evoked an E2-like pattern. Our data indicate a non-competitive inhibition of ATP hydrolysis that favors buildup of the E2-conformers of the enzyme. Chelerythrine as low as 0.5–1.5 lM resulted in an increase of intracellular Ca2+ on cultured PBMC cells. The inhibition of SERCA and the loss of cell Ca2+ homeostasis could in part be responsible for some described cytotoxic effects of the alkaloid. Thus, the choice of chelerythrine as a PKC-inhibitor should consider its potential cytotoxicity due to the alkaloid’s effects on SERCA. Ó 2015 Elsevier Inc. All rights reserved.
Introduction SERCA1 (sarco/endoplasmic reticulum Ca2+-ATPase), (E.C. 3.6.3.8.) is a P-type ATPase directly involved in cell Ca2+ responses and homeostasis. This enzyme translocates two Ca2+ ions from the cytoplasm into the endoplasmic reticulum using the energy derived from the hydrolysis of ATP, thereby contributing to decrease the amount of calcium in the cytoplasm. Thus, cell responses dependent on high
⇑ Corresponding authors at: Laboratório de Estrutura e Regulação de Proteínas e ATPases, Instituto de Bioquímica Médica Leopoldo de Meis, UFRJ, CCS, sala H2-026, Avenida Brigadeiro Trompowsky s/n° Ilha do Fundão, Rio de Janeiro, RJ 21941-590, Brazil. E-mail addresses: [email protected] (S.M. Vieira), [email protected]. br (C.F.L. Fontes), [email protected] (J.A. Mignaco). 1 Abbreviations used: SERCA, sarco/endoplasmic reticulum Ca2+-ATPase; pNPPase, pnitrophenylphosphatase; pNPP, p-nitrophenylphosphate; CHE, chelerythrine; DTT, DLdithiotreitol; PMSF, phenylmethylsulfonyl fluoride; MTT, 1-(4,5-dimethylthiazol-2yl)-3,5-diphenylformazan; SRV, Sarcoplasmic reticulum vesicles; PBMC, Peripheral Blood Mononuclear Cells; FBS, fetal bovine serum; PTP, permeability transition pore; PMCA, plasma membrane Ca2+-ATPase. http://dx.doi.org/10.1016/j.abb.2015.02.019 0003-9861/Ó 2015 Elsevier Inc. All rights reserved.
cytoplasmic calcium concentrations, like muscle contraction, are terminated. As for other P-type ATPases, SERCA’s catalytic cycle presents two main conformational states, E1 and E2, and the enzyme becomes reversibly phosphorylated at an aspartate (D351) within the DKTGTLT sequence common to all P-ATPases [1–4]. The phosphorylated enzyme (E1–P) occludes two calcium ions coordinated by polar and ionic side chains of aminoacid residues present at the transmembrane helices M4, M5, M6 and M8 [5] and after conversion to the E2–P form the calcium sites, now facing the luminal side of the reticulum, lose affinity and release the ion inside the ER. The enzyme is then dephosphorylated regenerating the free enzyme ready for a new cycle (Scheme 1) [6,7]. This dephosphorylation is the rate-limiting step of the cycle [8]. The dephosphorylation is controlled by the conserved TGES motif, which coordinate and activate one H2O molecule for nucleophilic attack by abstracting a proton [9]. It has been demonstrated that protonation of the residues at the empty Ca2+ binding sites in E2–P is mandatory for normal dephosphorylation of E2–P, and that mutations at the Ca2+ binding sites could affect this protonation [7,10]. Besides ATP, SERCA can hydrolyze pseudosubstrates, like p-nitrophenylphosphate (pNPP) [11,12] or
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preparation performed as [28] was chosen due to purity reasons. Protein concentrations were determined according to Lowry et al. [29] using bovine serum albumin as standard. pNPPase activity
Scheme 1. The simplified E1/E2 cycle of SERCA (Adapted from 6,7).
acetylphosphate [12,13], and their hydrolysis is also able to sustain Ca2+ transport [13,14]. SERCA, as other members of the P-type ATPase family, shows a biphasic kinetic behavior for the hydrolysis of ATP [15,16]. At micromolar ATP concentrations an increase of hydrolytic activity is observed and the phosphoenzyme builds up. The increase of ATP to millimolar concentrations leads to higher rates of ATP hydrolysis yet without a further increase on the E–P amount. This kinetic behavior for long time raised a debate about whether one or two ATP-binding sites existed for each SERCA monomer. Structural evidence supports the one-site hypothesis, and thus the second ATPase burst must result from a kinetic effect prompted by ATPbinding to the already phosphorylated catalytic portion [17,18]. Chelerythrine (CHE) is an isoquinoline alkaloid commonly found in plants from the families Papaveraceae, Fumariaceae and Rutaceae [19]. Chelerythrine and similar alkaloids are used in the Traditional Chinese Medicine and, since some years ago, in medicinal chemistry because of their ample biological activities. Chelerythrine is a well characterized, cell permeable and potent inhibitor of PKC [20]. As chelerythrine effects on isolated PKC isoforms are well established, many reports describe that, for several cell lineages, chelerythrine (and related alkaloids) elicit cell responses attributed mainly to the inhibition of PKC [20–24]. However, authors also report other effects of chelerythrine, such as DNA damage, antifungal, antibacterial and antitumor properties, which have no direct relation to the inhibition of PKC [20]. Indeed, several authors claim that many of the alkaloid’s effects could be due to the interaction of this compound with numerous cell proteins, therefore pointing for the need of better control and interpretation of experiments when using whole or leaky cells [25,26]. The main goal of the present study was to show that besides its well known effects on PKC, chelerythrine is also a powerful inhibitor of the SERCA activity in the same concentration range widely used for the inhibition of PKC. Therefore, SERCA inhibition by chelerythrine could be a primary cause of cytotoxicity in target cells by the resulting loss of the cell calcium homeostasis. Materials and methods Reagents EDTA, DL-dithiotreitol (DTT), EGTA, HEPES, ATP (sodium salt), phosphatidylcholine, phenylmethylsulfonyl fluoride (PMSF), C12E8, 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), A23187, Fura-2AM and TRIZMA-base were purchased from SIGMA (St. Louis, MO, USA). Chelerythrine chloride was from either SIGMA or Calbiochem with similar results. [32P]Pi was from the Instituto de Pesquisas Energéticas e Nucleares – IPEN (São Paulo, SP, Brazil). Enzyme preparation Sarcoplasmic reticulum vesicles (SRV) were prepared from rabbit hind leg skeletal muscle as described by [27] and stored in liquid nitrogen. In the case of proteolysis experiments, Meissner
p-Nitrophenylphosphatase (pNPPase) activity was measured at 37 °C in media containing 20 mM Tris–Cl (pH 7.5), 0.5 mM EGTA or 0.05 mM CaCl2, 120 mM KCl, 10 mM MgCl2, 20 lg mL 1 SRV protein, 10 lM A23187, variable chelerythrine concentrations, and 3 mM pNPP. After 20–40 min, the reactions (0.5 mL) were quenched with 0.1 mL of 0.3 N NaOH, and 0.4 mL of deionized water, p-nitrophenol was measured by its absorption at 425 nm, assuming an extinction coefficient of 1.1 104 M 1 cm 1. Effect of chelerythrine chloride on the Ca2+-ATPase activity The Ca2+-ATPase activity was assayed at 37 °C in media with 100 mM Tris–Cl (pH 7.5), 4 mM MgCl2, 80 mM KCl, 0.05 mM CaCl2 and 28 lg mL 1 SRV protein, and different concentrations of chelerythrine. The reaction was initiated by addition of 3 mM Na+-ATP and stopped after 3 min by addition of 100 lL of 1% SDS. Activity was determined by counting the amount of radioactive Pi released from [c-32P]ATP. [c-32P]ATP was synthesized accordingly to the method of [30], with modifications described in [31]. The contamination of the [c-32P]ATP with free 32Pi never exceeded the limit of 0.5–1.0%. Phosphoenzyme determinations SERCA was phosphorylated by ATP in media containing 20 mM Tris–Cl (pH 7.5), 120 mM KCl, 10 mM MgCl2, 0.05 mM CaCl2, 100 lg mL 1 SRV protein, and the chelerythrine concentrations indicated. The reaction was started with 10 lM [c-32P]ATP and quenched after 5 s with 1 volume of cold 0.4 mM PCA plus 1 mM Pi. For the phosphorylation by [32P]Pi, 100 lg mL 1 SRV protein in an E2-favoring reaction buffer (20 mM MES-pH 6.0-with 10 mM MgCl2, 2 mM EGTA, and the indicated chelerythrine concentrations) was incubated and 4 mM [32P]Pi were added. Reactions were quenched as above. Samples were filtered in 0.45 lm Millipore filters HAWP246 employing a Hoeffer vacuum system apparatus and washed 3 times with the quenching solution. The radioactivity remaining in the filter (specifically bound phosphoenzyme) was counted using a Packard Tri-carb 2600 Liquid Scintillation Counter. Digestion of SERCA by trypsin in presence of chelerythrine chloride proteolysis was done in 50 mM Tris–Cl (pH 7.5), and 2 mg mL 1 SERCA was preincubated for 5 min with 1, 10, 50 and 100 lM chelerythrine chloride or with either 5 mM ADP or 5 mM sodium orthovanadate (final volume of 100 lL). Digestion was performed with 12 lg mL 1 trypsin for 2 min at 37 °C. The reaction was stopped with 10 lL of Laemmli denaturing buffer (6, final 62.5 mM Tris–Cl pH 6.8, 2% SDS, 5% b-mercaptoethanol) and stored in ice. SDS–PAGE was performed accordingly [32], on 10–21% acrylamide continuous gradient gels. 80 lg of total protein were applied to each well and slab gels run for 4 h at 120 V and 80 mA. The gels were stained with Coomassie blue. Intramolecular cross-linking with glutaraldehyde Reaction buffer contained 30 mM MOPS/Tris (pH 8.5, 7.5 or 6.5), 0.2 M sucrose, 80 mM KCl, 0.05 mM CaCl2, chelerythrine from 0.5 to 100 lM. After pre-incubation with chelerythrine for 5 min, 0.2 mg mL 1 SRV were incubated with 0.3 mM glutaraldehyde for 90 min at room temperature as described by [33]. The cross-
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linking reaction was quenched with 1/6 volume of denaturing buffer (6, final 62.5 mM Tris–Cl pH 6.8, 2% SDS and 5% b-mercaptoethanol). SDS–PAGE was performed on 7.5% gel acrylamide. The gel was stained with Coomassie blue and scanned. Band densities were calculated with the ImageJ software. Collection of blood samples and separation of monocytes Twenty milliliters of blood samples were obtained from healthy volunteers using sodium heparin (Roche, Brazil) as anticoagulant. Peripheral Blood Mononuclear Cells (PBMC) were separated by density gradient through centrifugation for 30 min at 400 g using 100% Ficoll–Histopaque reagent (GE, USA). After separation, cells were washed three times with PBS buffer for 8 min at 220 g. Intracellular calcium measurements 1 106 PBMC cells/mL were incubated with 1.5 lM FURA-2 AM in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) for 40 min at room temperature in the dark. Then cells were washed twice with PBS and suspended in PBS with 1 mM CaCl2. The [Ca2+]i was quantified according to [34] using an FP-6300 spectrofluorometer (JASCO, Japan) set to kex = 340/362 nm, kem = 540 nm, Slitex = 10 nm and Slitem = 5 nm, with constant stirring at 37 °C. After 2 min a concentrated volume of chelerythrine was added and the fluorescence shift recorded. Results Incubation of SR vesicles with increasing amounts of chelerythrine resulted in a marked, progressive inhibition of the Ca2+ATPase activity when ATP was the substrate. This culminated in a maximal inhibition of the ATPase activity in presence of 200 lM chelerythrine, with an IC501.5 lM (Fig. 1). A small fraction of the total ATPase activity could not be inhibited. This residual activity was probably due to the existence of a Ca2+-insensitive, Mg2+-ATPase activity typical of the sarcoplasmic reticulum [35] that is resilient to classical P-type ATPase inhibitors, and seems also not to be affected by chelerythrine. The inhibition of SERCA was partially (ca. 30%) reversed by centrifugation and washing, and the addition of thiol reducers did not contribute to any further reversal, evidencing a strong but not covalent binding to the protein (data not shown).
Fig. 1. Inhibition of SERCA by chelerythrine. SRV were incubated in presence of the alkaloid, and the ATPase activity measured in presence of increasing concentrations of chelerythrine. The assay media contained 0.028 mg/mL of SRV protein and 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 150 and 200 lM of chelerythrine. The nominal control activity of the Ca2+-ATPase was 3.2 lmoles/mg/min and the plot was presented as relative activities in comparison with the activity of the control samples. Each condition represents the mean ± SE (n = 4).
Fig. 1 clearly points to the existence of two components with distinct affinities for the inhibition of SERCA ATPase activity by chelerythrine. Therefore, a Dixon plot was made in order to identify each component and their relative affinities. This result is shown in Fig. 2, where the two components were clearly distinguished and we could thus calculate that the affinity for binding differs by one order of magnitude, with Ki1 = 1.2 lM e Ki2 = 26 lM. The profile of the Dixon plot already suggested a non-competitive type of inhibition for both high and low affinity inhibitory components. This non-competitive character was also confirmed by the substrate dependence for ATPase activity in absence or presence of chelerythrine (Fig. 3). The kinetic parameters shown in Table 1 make evident that although the Vmax decreased for any region of the ATP dependence curve there is a negligible decrease of the affinity for ATP at the catalytic site and a sizeable but not important decrease of the affinity at the ‘‘regulatory’’ site. SERCA is known to support high rates of pNPP hydrolysis in presence of Ca2+, developing a cycle much alike the ATPase one and also supporting Ca2+ transport. On the other hand, in absence of Ca2+ the enzyme is also able to promote a non-productive, futile hydrolysis of pNPP solely by the E2 conformer [14]. Chelerythrine clearly inhibited the unproductive pNPP hydrolysis conducted by the E2 conformer in absence of Ca2+ (Fig. 4). Although also seeming to present two components, this inhibition impaired only ca. 50% of the initial activity with as much as 50 lM chelerythrine. Nevertheless, in presence of Ca2+ the alkaloid promoted an opposite, dual effect on the dose–response curve for chelerythrine of the pNPPase activity, activating the phosphatase with a peak at 5–10 lM chelerythrine and progressively decreasing the activity thereafter, although with a drastically shifted IC50 if compared to the values found for the ATPase activity (Fig. 4). In order to figure out where chelerythrine would bind to SERCA, and whether this binding induced any conformational selection, we used the chemical modification approach of intramolecular cross-linking between residues at the nucleotide site with glutaraldehyde described by [33]. This cross-linking alters the mobility of SERCA in SDS–PAGE from 110 kDa to a band with apparent M.R. 125 kDa. Chelerythrine within 0.5–10 lM, the range of the high affinity component for inhibition of the SERCA ATPase activity, did not substantially impair formation of the 125 kDa band at any pH tested (6.5/7.5/8.5). However, a small decrease of crosslinking which saturated around 5–10 lM chelerythrine, became evident when the pH shifted from 6.5 to 8.5. Distinctive protection against the cross-linking occurred only at pH 7.5 and 8.5 and once the concentration of chelerythrine increased towards the low affinity component for inhibition of the ATPase activity (Fig. 5A–D). It is noteworthy that this protection at higher chelerythrine was not evident at pH 6.5 (Fig. 5A and D), which may suggest a dependence either on the conformation of the enzyme or on the ionization state of chelerythrine, or both, for the low affinity binding of the alkaloid. As Ca2+-ATPase and Ca2+-pNPPase activities in principle follow a similar cycle, we measured the phosphoenzyme levels for the steady-state in presence of chelerythrine using ATP as substrate. With 10 lM ATP as substrate no significant changes on the phosphoenzyme level were observed until beyond 5 lM chelerythrine (Fig. 6) when the ATPase activity was inhibited over 60% (see Figs. 1 and 3), suggesting that the nucleotide binding and phosphoryl transfer steps were not much affected. However, with higher chelerythrine the phosphoenzyme was significantly impaired. This suggests that binding of a second chelerythrine molecule to SERCA might impede ATP binding and phosphoryl transfer to the enzyme by shifting the enzyme to the E2-form, incompetent to be phosphorylated by 10 lM ATP [36]. The stabilization of the E2-related conformer might also be provoked by the binding of a second molecule of chelerythrine with lower affinity (which seems
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Fig. 2. Dixon plot of the inhibition of SERCA by chelerythrine. SRV were incubated with increasing concentrations of the alkaloid: (A) 0.2; 0.5; 1; 2 and 5 lM; (B) 0.5, 10, 20, 50; 100; 150 and 200 lM both performed with two concentrations of substrate (1 or 3 mM Na+-ATP). The assay media contained 0.028 mg/mL of SRV in buffer 100 mM Tris– HCl pH 7.5; 80 mM KCl; 4 mM MgCl2, 0.05 mM CaCl2, at 37 °C. Representative Dixon plots were obtained by the means of three independent chelerythrine inhibition experiments, error bars mean SE.
Fig. 3. ATP-dependence of SERCA with chelerythrine. The ATPase activity was measured in absence (-d-) or presence of 1 lM chelerythrine (-s-). The assay media contained 0.020 mg/mL SRV protein and increasing concentrations of ATP ranging from 1 lM to 2 mM. The kinetic parameters were calculated with the best fit of a double-michaelian curve using the kinetic module of Sigmaplot 8.0. Results are mean ± SE (n = 3).
Table 1 Kinetic parameters for inhibition of SERCA by 1 lM chelerythrine.
Control Chelerythrine
Km1 (lM)
Vmax1 (nmol/ mg/min)
Km2 (lM)
Vmax2 (nmol/ mg/min)
0.49 ± 0.19 0.58 ± 0.34
143.3 ± 32.5 62.2 ± 8.0
397.4 ± 62.6 762.6 ± 57.0
1707.6 ± 72.3 1283.7 ± 33.8
to be corroborated by data obtained in Figs. 1, 2, 4 and 5), in which an inhibitory effect on phosphatase activity and in E–P accumulation is observed with chelerythrine concentrations from 10 to 50 lM. The proteolysis of SERCA by trypsin is a method capable of detecting changes in the conformation of the sarco/endoplasmic reticulum Ca2+-ATPase as a result of incubation with a series of ligands and conditions. Trypsin first cleaves SERCA at a highly susceptible site at Arg505 of the N-domain (T1) producing two major fragments A and B. Further cleavage of fragment A at Arg198 of the A-domain (T2) yields subfragments A1 and A2 [37,38]. The rate at which these fragments are generated and further degraded by
Fig. 4. Effects of chelerythrine on the pNPPase activity of SERCA. The pNPPase activity of SRV was measured in presence of increasing concentrations of the alkaloid, either in presence of 50 lM CaCl2 (-d-) or 1 mM EGTA (-s-). Results are mean ± SE (n = 3).
trypsin depends on the conformational state of SERCA. We therefore performed proteolysis in the presence of diverse ligands known to shift SERCA conformation [33,39] toward the E1 (ADP) or the E2 (orthovanadate or 2 mM Ca2+) state and compared the digestion pattern obtained as a result of increasing amounts of chelerythrine. The results obtained are highly suggestive that, in low concentrations (up to 10 lM), chelerythrine binding to SERCA does not contribute to shift the enzyme conformation to anything different from the E1 pattern; but increasing the alkaloid concentration from 10 to 100 lM changes the scenario and ends up by giving an E2-like proteolysis pattern (Fig. 7). Control experiments (not shown) confirmed that the proteolytic activity of the trypsin was not changed by the presence of orthovanadate, ADP or chelerythrine ranging from 1 to 100 lM. Hitherto kinetic and structural data indicated that the first chelerythrine binding component although not directly shifting the conformational state had serious implications in impairing ATP hydrolysis. Conversely, based on the glutaraldehyde cross-linking and on the proteolysis assays the low affinity binding component appeared to shift enzyme conformation to a possible E2-like state. Therefore we verified the consequence of chelerythrine on the backdoor phosphorylation of SERCA by inorganic phosphate, which
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Fig. 5. Cross-link formation in SERCA by glutaraldehyde. 0.2 mg/mL of SRV were mixed with the indicated concentrations of chelerythrine and after that samples were incubated with 0.3 mM glutaraldehyde for 90 min. The results at different pH’s are presented as (A) pH = 6.5, (B) pH = 7.5, (C) pH = 8.5, and (D) density of bands. The gel staining was Coomassie blue-R (panels A–C). In panel (D), the symbols are (-d-) pH = 8.5, (-s-) pH = 7.5 and (-.-) pH = 6.5. The relative density of band was calculated and normalized to the 125 kD band in the absence of chelerythrine, which was indexed as 100%.
Fig. 6. Inhibition of SERCA ATP-driven phosphorylation by chelerythrine. SRV were incubated in presence of the alkaloid, and the phosphoenzyme formed quantified. The assay media contained 0.028 mg/mL of SRV protein, 10 lM [c-32P]ATP (specific activity of 2 105 cpm/nmol) and 1, 2, 5, 10, 20 and 50 lM of chelerythrine. The reactions were started by addition of the substrate, and after 2 s quenched with 1 vol. of perchloric acid 0.4 M (final 0.2 M). Each condition represents the mean ± S.D. (n = 3).
is possible when the enzyme is incubated at slightly acidic pH and in absence of Ca2+. In this situation the enzyme is phosphorylated by Pi on the E2 conformation [17,40]. Not unexpectedly, we found that when phosphorylation was performed with Pi as substrate (from the E2 state), the phosphoenzyme formation was increased by chelerythrine at any concentration of the alkaloid, indicating an effect of the alkaloid, shifting the enzyme to an E2-like
Fig. 7. Proteolysis of SERCA in presence of chelerythrine. SERCA (1.0 mg/mL) was incubated with chelerythrine, ADP or sodium orthovanadate, and digested by trypsin (0.012 mg/mL) for 2 min at 37 °C at pH 7.5. Proteolysis was stopped by the addition of electrophoresis sample buffer and equal amounts (40 lg) of digested protein was applied at a gradient (10–21%) SDS–PAGE. Lanes: 1 – SERCA (Meissner) control, 2 – Meissner + trypsin, 3–6 – same as 2 with increasing concentrations of chelerythrine (1/10/50/100), 7 – 5 mM ADP and 8 – 5 mM orthovanadate.
conformation that facilitates phosphorylation by Pi, increasing the observed E–P levels (Fig. 8). Although the direct binding and the inhibition of SERCA by chelerythrine were clear, we still had no evidence that this inhibition
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Scheme 2. pH-dependent (acid–base) equilibrium between the charged (iminium ion) and the uncharged (pseudobase) form of chelerythrine. The reported pKR+ values of chelerythrine in aqueous solutions range from 7.5 to 9.2 (Vlcˇková et al., 2004).
Fig. 8. Accumulation of Pi-driven phosphoenzyme in the presence of chelerythrine. The phosphorylation by [32P]Pi (specific activity of 2 105 cpm/nmol) was performed with 100 lg/mL SRV protein in an E2-favoring reaction buffer (20 mM MES pH 6.0, with 10 mM MgCl2 and the indicated chelerythrine concentrations). The enzyme was incubated at 37 °C and the reaction was started by the addition of 4 mM [32P]Pi. Reactions were stopped after 1 min by the addition of 1 vol. of 0.4 M perchloric acid. Results are mean ± S.D. (n = 3).
would occur in whole cells and contribute to impair cell Ca2+ homeostasis. We then labeled quiescent fresh PBMC cells with the cytosolic Ca2+ sensor Fura2-AM and incubated them with chelerythrine between 0.5 and 1.5 lM, which are usually employed in PKC inhibition assays (reported IC50 for PKC is 0.6 lM). It was remarkable that almost immediately the [Ca2+]i started to increase steadily for several minutes after the addition of the low chelerythrine amounts (Fig. 9). In the time lapse acquired the [Ca2+]i was increased by P60% using chelerythrine concentrations that are elsewhere reported to cause cytotoxicity [41–43].
Discussion Chelerythrine is an isoquinoline alkaloid widely used as inhibitor of PKC and related calcium-mediated signal transduction in cells. The reported EC50 value for PKC inhibition by chelerythrine is around 0.6 lM, and its effects seem to be due to its binding to the catalytic domain of PKC promoting a non-competitive inhibition with respect to ATP, but with a competitive inhibition regarding to phosphate acceptors like histone H3s [20]. From the chemical point of view, chelerythrine can interconvert between a quaternary iminium form (cationic form) and
pseudobase/hydroxide adducts (neutral form), with a pKa ranging 7.5–9.2 (Scheme 2) [44–46]. At physiological pH apparently chelerythrine permeates the cell membrane in the form of a nonpolar pseudobase [47,48]. Slaninová et al. [48] reported that chelerythrine enters the cell in the form of yellow-orange vesicles, indicating that the quaternary adduct form of the alkaloid enters the cell by pinocytosis. Once inside the cell, chelerythrine distributes within cell compartments [48,49]. The iminium bond of the quaternary form (C6@N+) is susceptible to nucleophilic attack, and this reaction is essential to the effect of the reported inhibition of SH-proteins [50]. In our experiments, performed at pH 7.5, we did not find any evidence pointing to the reaction of thiol groups of SERCA with chelerythrine. Isoquinoline alkaloids have been used in a wide range of applications, ranging from toothpaste additives as bacteriostatic agents [50] to potentially antitumor drugs [41,51–53]. Several reports describing pro-apoptotic effects of these alkaloids were published, and most of them implicate mitochondrial function alterations as the main causative factor of apoptosis [42,54,55]. A possible direct effect of chelerythrine in membrane ion-transporting ATPases has, though, been underestimated in the literature, with some older reports of sanguinarine and other alkaloids inhibiting Na,KATPase [56,57]. A recent paper describes the inhibitory effect of sanguinarine and its dihydroderivative on the Na,K-ATPase [58], and these authors claimed that the E1-form has the sanguinarine binding site in a more accessible state than the E2-form of the enzyme. We show in this work that chelerythrine, in concentrations very near to those used to inhibit PKC, strikingly impaired SERCA’s activity. SERCA inhibition could lead to a deregulated Ca2+ homeostasis in cells treated with the alkaloid and could at least in part be responsible for some of the reported effects of chelerythrine in cell cultures [42,54,59]. The mechanism by which chelerythrine inhibits SERCA is still not completely defined. SERCA is well characterized as presenting
Fig. 9. Intracellular increase of calcium in presence of chelerythrine. After incubation of PBMC with FURA-2 AM, cells were washed and resuspended with PBS and 1 mM CaCl2. Arrow indicates addition of chelerythrine after 2 min of fluorescence analysis in several chelerythrine conditions 0, 0.5, 1 and 1.5 lM. The asterisk represents the [Ca2+]i after exposition to 50 nM thapsigargin during 30 min as a control. In the case of absence of CHE, an equivalent amount of water was added during fluorescence measurements. (B) [Ca2+]i at 30 min, comparison between chelerythrine and thapsigargin. The results are representative of five separate experiments.
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two Km’s for ATP [15,16]. Therefore, it was of interest to gather whether the inhibition of the ATPase activity was competitive with the binding of ATP, since the inhibition of PKC by chelerythrine was already described to be non-competitive [20]. The ATP-dependence and kinetic parameters for the Ca2+-ATPase activity inhibited by chelerythrine showed that Km1 and Km2 were not significantly shifted, and the Dixon plot confirmed the non-competitive character of SERCA’s ATPase activity. Thus, our data support that low concentrations of chelerythrine inhibit SERCA and decrease the overall hydrolysis rate by a high affinity, non-competitive binding mechanism. Similarly, pNPP hydrolysis by the E2 conformer of the enzyme is also impaired at low chelerythrine concentrations, suggesting that phosphorylation/dephosphorylation steps in the E2 form might be slower. A conflicting result is obtained for pNPP hydrolysis in presence of Ca2+, where a slight, but consistent activation is seen with the same low chelerythrine concentrations. On the other hand, at low chelerythrine the SERCA phosphoenzyme levels formed with ATP were not altered, evidencing that, for instance, ATP binds and phosphorylates the enzyme, but further reaction steps are impaired. And as well, phosphorylation by Pi is enhanced with a biphasic profile. Therefore, the profile of all the activity curves and phosphorylation results altogether support that SERCA apparently presents two binding sites for the alkaloid. However, probably the kinetic effects observed depend on the rate-limiting step and on the enzyme conformers involved in the hydrolysis cycle for the different substrates and, for instance, also on the conformational changes involved when Ca2+ is transported. The consequences of chelerythrine binding to SERCA on the intramolecular cross-linking and on the digestion pattern by trypsin are suggestive about chelerythrine-induced conformational changes of the enzyme. As shown in Figs. 5 and 7, low chelerythrine does not seem to majorly prevent cross-linking or to modify the trypsin digestion pattern. Moreover, concentrations of the alkaloid above 10 lM result in gradual impairment of cross-linking and also result in an E2-like digestion pattern. Therefore, at least for chelerythrine concentrations corresponding to the 2nd, low affinity binding component that inhibit the ATP activity (apart from other kinetic effects), it is possible to suggest that chelerythrine induced an enzyme conformational drift to the E2 state. This proposal is also supported by the effects of high concentrations of chelerythrine inhibiting the ATP-driven phosphorylation and promoting Pi-driven phosphoenzyme accumulation (Figs. 6 and 8). The involvement of Ca2+ signaling in cell death is well recognized. Persistent high intracellular Ca2+ leads to mitochondrial Ca2+ overload, which in turn leads to opening of the mitochondrial permeability transition pore (PTP), dissipation of electrochemical gradients, mitochondrial swelling, and release of cytochrome c and other apoptotic signals, and ultimately leads to cell death [60,61]. The induction of apoptosis by inhibition of SERCA with thapsigargin, leading to Ca2+ stress, is documented [61] and results in an outcome comparable to the described for chelerythrine or the related alkaloid sanguinarine. Independent of the mechanism involved in the inhibition of SERCA, there is a close proximity between the concentrations of chelerythrine sufficient to impair the enzyme catalytic function and those which cause the rapid and continued increase in PBMC cytosolic calcium (see Fig. 9). Therefore, our results are suggestive of the direct involvement of SERCA inhibition by chelerythrine in triggering the increase in cell calcium that will, for instance, lead to apoptosis and cell death, which was reported employing diverse cell models [41,42,59]. Our observations do not rule out that besides SERCA other transporters involved in cell Ca2+ homeostasis could be (directly or indirectly) affected by chelerythrine. For instance, the plasma membrane Ca2+-ATPase (PMCA), a member of the P-type ATPase family that pumps Ca2+ outward the cell [62], presents a high
degree of structural and functional homology with both SERCA and Na, K-ATPase, and could therefore be a potential target of the alkaloid. Preliminary results show that the PMCA-driven ATP hydrolysis is in fact impaired by chelerythrine, and this is a subject of current investigation in our laboratory. In addition, the outward Ca2+ pumping capacity of the Na+/Ca2+ exchanger [62] could be affected by a slight, but consistent inhibition of the Na,K-ATPase by chelerythrine (roughly 20% inhibition with 2 lM of the alkaloid, not shown) that could also change the Na+ balance in the long term. No matter the fractional contribution of each mentioned mechanism, the outcome shall be the same: increased cell calcium triggering cell death. Our work raises the concern that, besides the inhibition of PKC by chelerythrine, that certainly will be of serious consequence to cells, a direct and relevant inhibition of SERCA will result in an uncontrolled cell calcium increase thereby triggering mitochondrial events that will ultimately lead to cell death by apoptosis in either healthy or disease-model cell types. Since the high affinity Ki observed for the chelerythrine-dependent inhibition of ATP hydrolysis by SERCA was 1.2 lM, which is only two times greater than the reported Ic50 of 0.66 lM for PKC inhibition, the safety for the use of chelerythrine as a specific pharmacological inhibitor of PKC is at least questionable. We suggest that more careful attention should be taken when testing chelerythrine and related alkaloids in mammalian cells, since chelerythrine is widely marketed and employed as a specific PKC inhibitor during signal-transduction based studies. Acknowledgments The helpful technical assistance of M.Sc. Mônica M. Freire and Geiza L. Fraga is greatly acknowledged. We also thank the formerly undergraduate students Ana Paula Carvalho and Catarina C. P. Prima for their contribution in the beginning of this project. A.P.C. and C.C.P.P. were recipients of undergraduate fellowships from CNPq. V.H.O. and O.C.M. were recipients of graduate fellowships from CNPq. C.F.L.F. is recipient of a research fellowship from CNPq. This work was supported by grants from CNPq, CAPES and FAPERJ. References [1] K.B. Axelsen, M.G. Palmgren, J. Mol. Evol. 46 (1998) 84–101. [2] J.V. Moller, B. Juul, M. le Maire, Biochim. Biophys. Acta 1286 (1996) 1–51. [3] J.P. Andersen, B. Vilsen, H. Nielsen, J.V. Møller, Biochemistry 25 (1986) 6439– 6447. [4] E. Carafoli, Annu. Rev. Physiol. 53 (1991) 531–547. [5] C. Toyoshima, M. Nakasako, H. Nomura, H. Ogawa, Nature 405 (2000) 647–655. [6] L. de Meis, A.L. Vianna, Annu. Rev. Biochem. 48 (1979) 275–292. [7] A.G. Lee, J.G. East, Biochem. J. 356 (2001) 665–683. [8] D. Lewis, R. Pilankatta, G. Inesi, G. Bartolommei, M.R. Moncelli, F. TadiniBuoninsegni, J. Biol. Chem. 287 (2012) 32717–32727. [9] C. Olesen, T.L. Sorensen, R.C. Nielsen, J.V. Moller, P. Nissen, Science 306 (2004) 2251–2255. [10] J.P. Andersen, Biosci. Rep. 15 (1995) 243–261. [11] J.M. Ribeiro, E.S. Aragão, A.L. Vianna, An. Acad. Bras. Cienc. 52 (1980) 403–409. [12] M. Ushimaru, Y. Fukushima, Biochem. Biophys. Res. Co. 353 (2007) 799–804. [13] L. Meis, W. Hasselbach, J. Biol. Chem. 246 (1971) 4759–4763. [14] J.A. Mignaco, O.H. Lupi, F.T. Santos, H. Barrabin, H.M. Scofano, Biochemistry 35 (1996) 3886–3891. [15] S. Verjovski-Almeida, G. Inesi, Biochim. Biophys. Acta 558 (1979) 119–125. [16] J.S. Taylor, D. Hattan, J. Biol. Chem. 254 (1979) 4402–4407. [17] P. Champeil, S. Riollet, S. Orlowski, F. Guillain, C.J. Seebregts, D.B. McIntosh, J. Biol. Chem. 263 (1988) 12288–12294. [18] J.M. Autry, J.M. Rubin, B. Svensson, J. Li, D.D. Thomas, J. Biol. Chem. 287 (2012) 39070–39082. [19] M. Wink (Ed.), Alkaloids. Biochemistry Ecology and Medical Applications, Plenum Press, London, 1998. pp. 265–298. [20] J.M. Herbert, J.M. Augereau, J. Gleye, J.P. Maffrand, Biochem. Biophys. Res. Commun. 172 (1990) 993–999. [21] S.J. Chmura, M.E. Dolan, A. Cha, H.J. Mauceri, D.W. Kufe, R.R. Weichselbaum, Clin. Cancer Res. 6 (2000) 737–742. [22] A. Shrivastava, Indian J. Exp. Biol. 45 (2007) 755–763.
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