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Abbreviations DCF-DA, 2’, 7’-dichlorodihydrofluorescein diacetate dye; 2-PTS, sodium 2-propenyl thiosulfate; ESI, electrospray ionization; GSH, reduced glutathione; GSSG, oxidized glutat

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Implications in apoptosis induction

Renato Sabelli1, Egidio Iorio2, Angelo De Martino3, Franca Podo2, Alessandro Ricci2,

Giuditta Viticchie`1, Giuseppe Rotilio3, Maurizio Paci1and Sonia Melino1

1 Department of Sciences and Chemical Technologies, University of Rome ‘‘Tor Vergata’’, Italy

2 Department of Cellular Biology and Neurosciences, Istituto Superiore di Sanita`, Rome, Italy

3 Department of Biological Sciences, University of Rome ‘‘Tor Vergata’’, Italy

The induction of programmed cell death by sulfane

sulfur compounds [alk(en)yl thiosulfate,

selenodigluta-thione, allyl disulfide, etc.] poses significant questions

concerning their metabolism and on the role of the

enzymes involved in the cancerogenesis Toohey [1]

suggested that uncontrolled proliferation of neoplastic cells is a result of sulfane sulfur deficiency or overacti-vity of the enzymes involved in their metabolism Several organosulfur compounds (OSCs) such as diallyl disulfide, diallyl trisulfide, allicin and (more

Keywords

garlic; mobile lipids; sodium 2-propenyl

thiosulfate (2-PTS); sulfane sulfur;

sulfurtransferase

Correspondence

S Melino, Dipartimento di Scienze e

Tecnologie Chimiche, University of Rome

‘‘Tor Vergata’’, via della Ricerca Scientifica

00133-Rome, Italy

Fax: +39 0672594328

Tel: +39 0672594449

E-mail: melinos@uniroma2.it

E lorio, Department of Cellular Biology and

Neurosciences, Istituto Superiore di Sanita´,

Viale Regina Elena, 299 00161 Rome, Italy

Fax: +39 06 49387143

Tel: +39 06 49902548

E-mail: egidio.iorio@iss.it

(Received 7 March 2008, revised 30 May

2008, accepted 3 June 2008)

doi:10.1111/j.1742-4658.2008.06535.x

Sodium 2-propenyl thiosulfate, a water-soluble organo-sulfane sulfur com-pound isolated from garlic, induces apoptosis in a number of cancer cells The molecular mechanism of action of sodium 2-propenyl thiosulfate has not been completely clarified In this work we investigated, by in vivo and

in vitro experiments, the effects of this compound on the expression and activity of rhodanese Rhodanese is a protein belonging to a family of enzymes widely present in all phyla and reputed to play a number of dis-tinct biological roles, such as cyanide detoxification, regeneration of iron– sulfur clusters and metabolism of sulfur sulfane compounds The cytotoxic effects of sodium 2-propenyl thiosulfate on HuT 78 cells were evaluated by flow cytometry and DNA fragmentation and by monitoring the progressive formation of mobile lipids by NMR spectroscopy Sodium 2-propenyl thio-sulfate was also found to induce inhibition of the sulfurtransferase activity

in tumor cells Interestingly, in vitro experiments using fluorescence spec-troscopy, kinetic studies and MS analysis showed that sodium 2-propenyl thiosulfate was able to bind the sulfur-free form of the rhodanese, inhibit-ing its thiosulfate:cyanide-sulfurtransferase activity by thiolation of the catalytic cysteine The activity of the enzyme was restored by thioredoxin

in a concentration-dependent and time-dependent manner Our results sug-gest an important involvement of the essential thioredoxin–thioredoxin reductase system in cancer cell cytotoxicity by organo-sulfane sulfur com-pounds and highlight the correlation between apoptosis induced by these compounds and the damage to the mitochondrial enzymes involved in the repair of the Fe–S cluster and in the detoxification system

Abbreviations

DCF-DA, 2’, 7’-dichlorodihydrofluorescein diacetate dye; 2-PTS, sodium 2-propenyl thiosulfate; ESI, electrospray ionization; GSH, reduced glutathione; GSSG, oxidized glutathione; OSC, organosulfur compound; PCho, phosphocholine; RhdA(E), sulfur-free form of rhodanese; RhdA(ES), sulfur-loaded rhodanese; RhdA, recombinant rhodanese from Azotobacter vinelandii; RhdA-PS, propexylsulfide-form of rhodanese; ROS, reactive oxygen species; Trd, thioredoxin reductase; Trx, thioredoxin; TST, thiosulfate:cyanide sulfurtransferase.

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recently), n-propyl thiosulfate and sodium 2-propenyl

thiosulfate (2-PTS) have been shown to suppress the

proliferation of tumor cells in vitro through the

induc-tion of apoptosis [2–7] The biochemical mechanisms

underlying the antitumorigenic and antiproliferative

effects of garlic-derived OSCs are not yet fully

under-stood, although it seems likely that the rate of

clear-ance of allyl sulfur groups from cells is a determinant

of the overall response It has also been hypothesized

that a reduced or incorrect functionality of enzymes

involved in the metabolism of OSCs may cause an

excess of seleno or sulfane sulfur compounds in the

cell, inducing the onset of degenerative states and

apoptosis The in vitro study of their interaction with

enzymes probably involved in the metabolism of sulfur

may explain their in vivo effects Sulfurtransferases,

enzymes that act at the borderline between sulfur and

selenium metabolism, may have an important role in

the processes induced by these sulfur compounds In

particular, rhodanese displays a sulfurtransferase

activ-ity in vitro, transferring the sulfane sulfur atom from

thiosulfate to cyanide to produce thiocyanate by a

double-displacement mechanism [thiosulfate:cyanide

sulfurtransferase EC 2.8.1.1, (TST)] [8] In this activity,

rhodanese cycles between two stable intermediates,

namely a sulfur-loaded form (ES) and a sulfur-free

form (E) The observed abundance of potentially

func-tional rhodanese-like proteins suggests that members

of this homology superfamily (accession number:

PF00581; http://sanger.ac.uk/cgi-bin/Pfam) may play

distinct biological roles [9,10] Rhodanese modules,

either alone or in combination with other proteins, can

perform a variety of roles, ranging from transport

mechanisms of sulfur⁄ selenium in a biologically

avail-able form [11–13], to the modulation of general

detoxification processes [8] and to the restoration of

iron–sulfur centres in Fe–S proteins, such as ferredoxin

[14] In the last few years, many different studies have

supported the hypothesis that the rhodanese-like

pro-teins have roles in ‘managing’ stress tolerance and in

maintaining redox homeostasis [15–18] It is also

note-worthy that a study, using microarray to examine the

gene expression in colonic mucosa from cancerous and

normal tissues, hypothesized that a possible cause of

colorectal cancer carcinogenesis might be located in

the mitochondria and identified the rhodanese gene as

one out of three mitochondrial genes that had a

statis-tically significant decrease in expression from normal

tissue to tumor at every Dukes’ stage A–D [19,20]

More recently an increase in TST expression was

observed in colonocyte differentiation In a human

colon cancer cell line, TST activity and expression were

significantly increased by butyrate and by

histone-deacetylase inhibitors, which promote the differentia-tion of these cells [21] Thus, butyrate could protect the colonocytes from sulfide-induced cytotoxicity, thus promoting TST expression [21] Down-regulation of rhodanese gene expression has also been observed in diseases such as Friedreich’s ataxia [22] The associa-tion between the expression of rhodanese and degener-ative states might make rhodanese a potential tumor⁄ disease biomarker and treatment target Not all cells are equally susceptible to the deleterious effects of the garlic sulfur compounds and, in particular, non-neoplastic cells are less susceptible [3,23] Therefore, the greater sensitivity of tumor cells to these com-pounds may be related to a down-regulated expression

of TST, which leads to a reduced rate of clearance of these compounds; notably, epidemiological investiga-tion has revealed that increased consumpinvestiga-tion of garlic diminishes the risk of stomach and colorectal cancers [24–27] To explore the importance of rhodanese in the metabolism of the allyl-sulfur compounds, we investi-gated, in the present work, the effect of some natural sulfur constituents of garlic on TST activity In particular, we studied the interaction of 2-PTS with rhodanese and investigated the effect of this OSC on the expression and activity of rhodanese Sodium 2-propenyl thiosulfate is present in aqueous garlic extract [28] and has an anti-aggregator effect in vitro

on both canine and human platelets as a result of the inhibition of cyclooxygenase activity [29] The anti-tumor effect of 2-PTS, resulting from induction of the apoptosis process, has been recently investigated [7] The data reported here show that 2-PTS is able to bind to the active site of rhodanese, resulting in TST inhibition We also investigated the role of reduced thioredoxin (Trx) as a possible sulfur acceptor in this reaction to restore TST activity Moreover, the cyto-toxic effect of 2-PTS on HuT 78 cells was also evalu-ated using flow cytometry analysis after treatment and

by monitoring the progressive formation of mobile lipids by NMR spectroscopy All results obtained provide evidence that highlights the possible role

of rhodanese in the management of the cytotoxicity

of reactive OSCs in tumor cells and may contribute to the design of a scheme of the mechanism of action of 2-PTS as an apoptosis inducer

Results

Inhibition of cell cycle progression and induction

of apoptosis by 2-PTS The effects of 2-PTS on the human T-lymphoblastoid cell line, HuT 78, were analysed and a typical

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time-dependent and dose-time-dependent inhibition of cell growth

of these cells as a result of the presence of 2-PTS was

observed In fact, a statistically significant decrease in

the number of viable cells, at 0.25, 0.5 and 1 mm

concen-trations of 2-PTS and at different time-points, was

found when comparing control and thiosulfate-treated

cells (data not shown) About 75 and 10% of the HuT

78 cells were viable following exposure to 0.5 mm 2-PTS

for 24 and 48 h respectively (Fig 1A) The

growth-inhibitory effects of 2-PTS were determined by using the

Trypan Blue dye-exclusion assay Flow cytometric

anal-ysis of HuT 78 cells after 24 and 48 h of treatment with

0.5 mm 2-PTS, following staining with propidium

iodide, resulted in a statistically significant increase in the fraction of subG1 compared with the control (Fig 1B), showing a characteristic feature of apoptosis Sodium 2-propenyl thiosulfate induced an increase in the fraction of HuT 78 cells in the G2⁄ M phase after

24 h of treatment, reaching a value of 67.43% (Fig 1B) After 48 h, the percentage of the cell population that showed apoptotic features (subG1 region) was 39%, suggesting that an antiproliferative activity of 2-PTS against HuT 78 cells (blockage in G2⁄ M phase) results

in the triggering of apoptotsis

The effect of treatment with 2-PTS on DNA frag-mentation was assessed by analysis of the agarose-gel electrophoretic pattern of HuT 78 cells (see supplemen-tary Fig S1) A typical DNA-fragmentation pattern was observed as early as at 24 h after the addition of 2-PTS, thus confirming the apoptosis-inducing effects of 2-PTS The role of reactive oxygen species (ROS) as potential mediators of 2-PTS-induced cytotoxicity in HuT 78 cells was also investigated Figure 1C shows the production

of ROS during the first 3 h of treatment with 2-PTS HuT 78 cells underwent an increase of ROS flux as early

as 1 h after addition of 2-PTS These results implied a strict association between 2-PTS effects and oxidative imbalance Reduced glutathione (GSH) is the most important physiological antioxidant, both by directly reacting with ROS and indirectly by preserving cysteine residues of proteins from irreversible oxidations, so giv-ing rise to GS-R The intracellular reduced (GSH) and oxidized (GSSG) glutathione levels were measured using HPLC chromatography in order to show the potential involvement of this redox system in the resistance of HuT 78 cells to treatment with 2-PTS Untreated cells showed an average intracellular GSH concentration of

 55 nmolÆmg)1of protein, whereas 2-PTS-treated cells showed a rapid and sustained increase of GSH levels (118.5 nmolÆmg)1 protein) up to 12 h, probably as a result of to the detoxification process The intracellular GSSG content was not significantly affected by treat-ment with 2-PTS and remained at very low levels

Formation of 1H-NMR-visible mobile lipids during 2-PTS-induced apoptosis

The apoptotic parameters were quantitatively moni-tored by NMR spectroscopy carried out on intact HuT

78 cells and on their aqueous cell extracts after cell expo-sure to 2-PTS Detection of the progressive formation of mobile lipids in intact cells indicated the induction of apoptosis after treatment with 2-PTS (from 6 to 48 h) Resonances centered at d = 1.3 p.p.m., as a result of the saturated fatty acyl chain methylene segments -(CH2)n-, and at d = 0.9 p.p.m., arising from methyl

A

B

C

Fig 1 In vitro effect of 2-PTS on the growth of HuT 78 cells (A)

Viability of HuT 78 cells in culture after treatment with 0.5 m M

2-PTS Trypan Blue staining was used to differentiate viable from

nonviable cells Data are expressed as mean ± SD *P < 0.005,

**P < 0.001 (n = 8) (B) Percentage of the cell cycle distribution of

HuT 78 cells after 24 and 48 h of treatment with 0.5 m M 2-PTS (C)

Intracellular production of ROS in HuT 78 cells after 1 and 3 h of

treatment with 0.5 m M 2-PTS, detected by measurement of DCF

fluorescence using a FACSCalibur flow cytometer Data are

expressed as mean ± SD *P < 0.001, **P < 0.05 (n = 3).

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groups, were followed Quantitative analysis of mobile

lipid spectral profiles (see the supplementary material)

was obtained by measuring the peak area (a) ratio

Rchains= a[(CH2)n]lip⁄ a[CH3]tot, according to our

previ-ous work [30], where a[(CH2)n]lip is the integral of the

mobile lipids, (CH2)nis the resonance and a(CH3)totis

the integral of the total CH3 resonance at 0.9 p.p.m

caused by amino acids and lipids The value Rchains in

untreated control cells was 0.20 ± 0.1, not significantly

different from that measured in HuT 78 cells exposed to

0.25 mm 2-PTS at any time-point The average Rchains

value at 24 h of exposure to 2-PTS increased with the

apoptotic fraction from 1.25 to 1.56 in cells treated with

0.5 and 1.0 mm 2-PTS, respectively The spectral profiles

of cells treated with either 0.5 or 1.0 mm 2-PTS for 48 h

were mainly characterized by signals from mobile lipids

(Rchains= 4.45 ± 1.1), while all resonances caused by

small aqueous metabolites decreased to very low levels

as a result of the massive apoptosis of treated HuT 78

cells, probably associated with loss of cell integrity

Quantitative analysis on 1H-NMR spectra of cell

extracts after exposure to 0.5 mm 2-PTS for an

interme-diate time interval (24 h, not yet associated with late

apoptosis) showed that the intracellular level of taurine

(measured from the triplet centred at d = 3.45 p.p.m.)

decreased about two-fold from a basal control level of

13.0 ± 2.4 to 6.5 ± 1.0 nmol per 106cells) Under the

same experimental conditions, individual

choline-containing metabolites (tCho, detected under a

 3.22 p.p.m.) underwent differential changes; in fact

phosphocholine (PCho) decreased by 45% (from

15.1 ± 1.4 to 8.1 ± 0.8 nmol per 106 cells), while free

choline and glycerophosphocholine both increased by

about three-fold The changes in the levels of

water-soluble choline-containing metabolites observed in cells

treated with 2-PTS for 24 h probably reflect the

progres-sive activation of phospholipases and

phosphodiester-ases On the other hand, a complex network of

pathways may, in principle, contribute to the measured

decrease in PCho, a metabolite particularly sensitive to

conditions determining a block in cell proliferation

and⁄ or to the activation of enzymes involved in choline–

phospholipid degradation In fact, both increases and

decreases in PCho have been reported to occur in

differ-ent systems of apoptotic induction, according to

partic-ular experimental conditions [30]

Effect of 2-PTS on TST expression and activity in

HuT 78 cells

The effects of 2-PTS on the expression and activity of

TST in HuT 78 cells were analyzed HuT 78 cells were

treated without and with 2-PTS at various concentra-tions (0.25, 0.5 and 1 mm) Figure 2A shows the western blot of the HuT 78 lysates after 8 and 24 h of treatment with 0.5 mm 2-PTS Densitometry measure-ments of western blots, corrected for actin or for glyceraldehyde-3-phosphate dehydrogenase (see the supplementary material) expression, show that no sig-nificant variation of the expression of TST with respect

to the control was induced by treatment with 2-PTS (see Fig 2A) By contrast, a reduction of TST activity was observed after 24 h, as shown in Fig 2B

Interaction of 2-PTS and allyl compounds with rhodanese

In vitro interaction and kinetic studies were performed using the recombinant Azotobacter vinelandii rhoda-nese (RhdA) [31–33] RhdA has similar properties and kinetic behaviour and a high structural homology with bovine rhodanese [34], which is the rhodanese consid-ered as an appropriate model for using to study human rhodanese [32,33,35] The A vinelandii

rhoda-A

B

Fig 2 Effect of 2-PTS on TST expression and activity in HuT 78 cells (A) Western immunoblotting showing the expression of TST

in HuT 78 cell lysates after treatment with 2-PTS Thirty micro-grams of protein lysates from untreated cells (CTRL) incubated for

8 and 24 h, and from cells treated with 0.5 m M 2-PTS for 8 and

24 h, were analysed A monoclonal anti-actin Ig was used as a con-trol of the protein concentrations; and densitometry measurements

of western immunoblotting were calculated by comparison with the intensity of actin expression (B) Percentage the TST activity of HuT 78 cellular extracts [untreated cells (CTRL) and cells treated with 0.5 m M 2-PTS (2-PTS) after 8, 24 and 48 h of incubation] The control after 24 h was used as the reference value *P < 0.05.

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nese has been shown to contain only one cysteine

resi-due, which is essential for the catalytic reaction, and

therefore it represents a good model for using to study

the possible interaction of TST proteins with other

proteins or molecules involved in sulfur and selenium

metabolism [35,36]

In order to investigate the action of 2-PTS on

sulfur-transferase activity, we analyzed the effect of 2-PTS by

monitoring the changes of intrinsic fluorescence that

occur when the rhodanese cycles between the

sulfur-free [RhdA(E)] form and the sulfur-loaded [RhdA(ES)]

form and that are caused by long-range energy

trans-fer and local conformational changes in the protein

[36–40]

No fluorescence changes were observed when 2-PTS

was added to RhdA(ES) (Fig 3A) By contrast, the

addition of 2-PTS induced an evident quenching of

intrinsic fluorescence of RhdA(E) (Fig 3B), in a

con-centration-dependent manner, indicating a specific

interaction of 2-PTS with the active site Moreover,

the quantum yield of intrinsic fluorescence of RhdA(E)

after the addition of 2-PTS was lower than that

obtained following the addition of thiosulfate

(DF% = 27.2 compared with thiosulfate DF% =

15.7) (Fig 3B) This major fluorescence quenching was

probably a result of the closeness of the allyl group to

the Trp residues present in the protein active site, and

is probably responsible for the differential quenching

of the intrinsic fluorescence in the two states of the

enzyme [37,39,41] Interestingly, cyanide did not

restore the unloaded form of the enzyme (Fig 3C) and

a significant loss of the TST activity of the enzyme

was observed

Inactivation of sulfur-free rhodanese by 2-PTS

and characterization of the derivative protein

In order to study chemical modifications of sulfur-free

rhodanese, it is necessary first to remove the persulfide

from the enzyme by adding an excess of cyanide to the

protein, thus forming the thiocyanate product and

sulfur-free rhodanese As sulfur-free rhodanese is

somewhat unstable, it is customary to add the

modify-ing reagent immediately after cyanide treatment We

performed the experiment in this way to analyze the

effect of the 2-PTS on the sulfur-free form of the

enzyme Figure 4 shows the time course of inactivation

of RhdA(E) caused by an interaction with 2-PTS

Pre-incubation of RhdA in the presence of a three-fold

molar excess of cyanide and a 200-fold molar excess of

2-PTS induced a decrease of sulfurtransferase activity

over time, and a complete loss of sulfurtransferase

activity was observed after 90 min at 37C (Fig 4A)

The activity of treated RhdA was not restored after dialysis, indicating that stable binding occurs between 2-PTS and RhdA(E) By contrast, no inhibition of the TST activity was observed after pre-incubation of RhdA(ES) in the presence of the same concentration

of 2-PTS without CN- TST activity of the propenyl-sulfide-form of rhodanese, RhdA-PS, was restored by treatment with dithiothreitol In fact, 53.6% of the TST activity, with respect to the TST activity of

A

B

C

Fig 3 Intrinsic fluorescence changes of RhdA following the addi-tion of substrates (A) RhdA(ES) (2.3 l M ) ( ), after the addition of

460 l M 2-PTS (- -) and after the addition of 460 l M CN - (- - -); (B) RhdA(E) (4.6 l M ) ( _ ), with a molar ratio E: thiosulfate 1 : 400 (- - - -), with a molar ratio E:2-PTS 1 : 200 (_ _ _) and E:2-PTS

1 : 400 ( - ); and (C) RhdA(E) (5 l M ) ( _ ), in the presence of 2-PTS (E: 2-PTS 1 : 250 c ⁄ c) ( _ _ _ ), E: 2-PTS 1 : 500 c ⁄ c (- -) and after the addition of CN-(E: 2-PTS: CN 1 : 500 : 1000 c ⁄ c ⁄ c) (- - - -) a.u., arbitrary units.

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RhdA(ES) before treatment, was recovered after

30 min of incubation at room temperature (23C) (see

Fig 4B) These results were in agreement with an

oxi-dation state of the catalytic Cys Thiosulfate

sulfur-transferase activity of RhdA-PS was also restored

when Trx protein was added to the solution and this

activity was dependent on the Trx concentration

(Fig 5A) Thioredoxin was able to re-establish 66% of

the TST activity of RhdA-PS when was incubated with

Trx at an RhdA-PS⁄ Trx molar ratio of 1 : 2 The yield

of the recovery was faster and higher when thioredoxin

reductase (Trd) and NADPH were also present in the

solution Figure 5B shows the recovery of the TST

activity of RhdA-PS in the presence of Trd, NADPH,

and different molar concentrations of reduced Trx A

total recovery of the TST activity was obtained after

70 min of incubation with 0.5 lm Trx No increase of thiocyanate production was observed in the presence

of Trx, Trd and NADPH without rhodanese These results, together with the fluorescence spectra, indicate

an interaction of 2-PTS with the catalytic Cys Thus, either the reduced Trx or dithiothreitol can reduce a disulfide bond between the OSC and the catalytic cys-teine of RhdA, and thereby restore the enzyme to its active state This hypothesis was confirmed by MS analysis of the new form of the enzyme RP-HPLC chromatography of RhdA-PS and RhdA(E) was per-formed, followed by electrospray ionization (ESI) MS analysis Although the two forms of the protein showed the same retention times in RP-HPLC (see the supplementary material), they showed different molec-ular mass peaks In fact, RhdA-PS and RhdA(E) had

m⁄ z 31138.9 ± 3.19 and m ⁄ z 31063.8 ± 3, respec-tively These results are consistent with thiolation of

A

B

Fig 4 Inhibition of the sulfur-free form of RhdA by 2-PTS (A)

Time-dependent decrease of the TST activity of 48.3 l M RhdA(E) in

50 m M Tris–HCl buffer, pH 8.0, 0.3 M NaCl RhdA(E) was treated

with a three-fold molar excess of cyanide and a 200-fold molar

excess of 2-PTS at 37 C (B) Effects of dithiothreitol on the TST

activity of RhdA(ES) and RhdA-PS The proteins were incubated

with 4 m M dithiothreitol at room temperature and the TST activity

was assayed after 0, 15 and 30 min RhdA(ES) and RhdA-PS were

treated identically, except that cyanide and 2-PTS were absent

dur-ing the treatment of RhdA(ES) The TST activity of the enzyme

before treatment was taken to represent 100% activity Each value

represents the average of three independent determinations DTT,

dithiothreitol.

A

B

Fig 5 Recovery of the TST activity of 17 l M RhdA-PS detected using the So¨rbo assay (A) Recovery of TST activity after 2 h of incubation at 25 C in the absence and in the presence of thiore-doxin at molar ratios 1 : 0, 1 : 0.5, 1 : 1 and 1 : 2 c ⁄ c RhdA-PS ⁄ Trx All values are expressed as a percentage of the TST activity value

of RhdA(ES) (B) Recovery of the TST activity of 8.1 l M RhdA-PS after incubation in the presence of 0.1 U Trd, 50 l M NADPH and different concentrations of Trx (0, 0.05, 0.15, 0.25 and 0.5 l M ).

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the catalytic cysteine of the enzyme with a

propenylsul-fide (m⁄ z 73), confirming modification of the protein at

the catalytic site

Sensitivity of RhdA-PS to proteolysis

To characterize the RhdA-PS form in greater detail,

limited proteolysis of RhdA-PS was performed

Lim-ited proteolysis of globular proteins generally occurs at

sites that contain the most flexible regions of the

poly-peptide chain within a domain or at the flexible hinges

between domains Therefore, a limited trypsin

diges-tion of RhdA-PS was performed to investigate the

flex-ibility of this modified enzyme As previously

observed, the sulfur-loaded form of RhdA [RhdA(ES)]

[42] appeared to be quite resistant to limited

proteoly-sis In fact, Rhd(ES), which was treated in the same

manner as for RhdA-PS, but in the absence of cyanide

and 2-PTS, was not proteolyzed by trypsin and

remained intact, even after incubation overnight

(Fig 6A) By contrast, RhdA-PS showed a higher

sen-sitivity to proteolysis than RhdA(ES), as shown in

Fig 6B,and a band rapid digestion was observed A

stable daughter band (b band), of about 17.3 kDa,

appeared after a few minutes of proteolysis and was

present also after many hours of digestion These data

suggest that RhdA-PS is more flexible than RhdA(ES),

probably as a result of local conformational

differ-ences RhdA-PS showed behaviour very similar to that

previously observed for the alkylated and oxidated

forms of the bovine rhodanese [43,44] In fact, limited

proteolysis of the alkylated and oxidized forms yielded

fragments that were about half of the apparent

molec-ular mass of the protein, as a result of cleavage at the

interdomain tether that connects the two domains into

which the single polypeptide chain protein is folded

[42,43,45] Thus, the inactivation of the rhodanese by

2-PTS induces local conformational changes that make

the enzyme much more sensitive to proteolytic

degra-dation

RhdA-PS form catalysed the Trx oxidation

Thioredoxin oxidation analyses were performed with

the aim of clarifying the mechanism involved in

restor-ing the TST activity of RhdA-PS The oxidation of

Trx by RhdA was observed by NADPH oxidation

The change in NADPH concentration was caused by

oxidation of the reduced Trx to its disulfide form,

which was reduced by NADPH in the reaction

cata-lyzed by Trd The presence of RhdA(ES) at an

equi-molar concentration of Trx caused oxidation of the

reduced Trx at equilibrium with NADPH, as

previ-ously observed for the bovine rhodanese [46,47] even

in the absence of a sulfur donor (Fig 7A) Thus, also

in this case, reduced Trx behaves as sulfur-acceptor substrate Control experiments showed that no oxida-tion of NADPH occurred in the presence of rhodanese when reduced Trx was absent The addition of 2-PTS (as a substrate of RhdA) to the solution resulted in further oxidation of NADPH (Fig 7A) 2-PTS was also able to oxidize Trx, but the presence of RhdA caused an increase in the NADPH oxidation rate (Fig 7A) In addition, RhdA-PS was able to catalyze Trx oxidation A rapid decrease in the concentration

A

B

Fig 6 Time course of trypsin digestion of RhdA-PS and RhdA(ES) Three-hundred micrograms of enzyme was subjected to limited digestion with 1% (w ⁄ w) trypsin in 1 mL of 50 m M Tris–HCl buffer,

pH 8, at room temperature After the reaction the samples were subjected to SDS-PAGE (A) Lanes 2–7, proteolysis products of RhdA(ES) (lane 1) at 0, 5, 10, 15, 20 and 30 min and overnight incu-bation (B) Lanes 2–8, proteolysis products of RhdA-PS (lane 1) at

0, 5, 10, 15, 20, 30, 60 min and overnight incubation, respectively.

‘a’ and ‘b’ bands are the parent and daughter bands, at about 29.7 and 17.3 kDa respectively Molecular markers are on the left STDs, molecular mass standards.

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concentration of RhdA-PS was added to the stabilized

solution containing Trx, 0.1 U Trd and 50 lm NADPH

(Fig 7B) Moreover, the subsequent addition of 2-PTS

to the solution led to a further increase in NADPH

oxidation These results indicate that the rhodanese–

Trx–Trd system is not inhibited by 2-PTS and that this

system has an antioxidant action against OSCs as well

a sulfane sulfur detoxification system

Discussion

Natural compounds, which improve detoxification

enzymes and⁄ or reduce the expression and activity of

the carcinogen activating enzymes, are good candidates

for cancer chemoprevention The controlled

prolifera-tion of the cell may be a result of the presence of a

sul-fane sulfur compound that has the ability to reduce or

promote the activity of important proteins implicated

in the cellular process Many OSCs present in allium vegetables have been shown to be able to inhibit the proliferation of cancer cells [4,48–51] The induction of programmed cell death by sulfane sulfur compounds raises relevant questions about the role of enzymes involved in their metabolism A feature of a neoplastic cell is the residual activity of cysteine aminotransfer-ase, 3-mercaptopyruvate sulfurtransferase and rhoda-nese, as well as the total lack of cystathionase activity [52]; in fact, the biosynthesis and transport of com-pounds from the sulfane sulfur pool does not occur in these cells [53]

Our studies presented here, on HuT 78 cells, showed that the viability of these cells is reduced significantly upon 24 h of exposure to 2-PTS and that this reduced growth rate was related to a blockage in the G2⁄ M phase of the cell cycle

The detection, by NMR, of increasing amounts of mobile lipids in 2-PTS-treated HuT 78 cells, further supports mitochondrial dysfunction in these cells In fact, several studies have reported the appearance of mobile lipids in a variety of cells induced to apoptosis [33,54–56], in which the characteristic apoptotic pheno-type generally results in loss of the mitochondrial membrane potential, release of cytochrome c and mito-chondrial-dependent activation of effector caspases Recently, alterations of mitochondrial functions by different uncouplers of the respiratory chain have been found to be responsible for the accumulation of intra-cellular lipid bodies and therefore for the detection of NMR-visible mobile lipids in intact HuT 78 cells (E lorio, C Testa, A Stringaro, M Condello, G Ara-ncia, E Lococo, R Carnevale, R Strom, L Lenti &

F Podo, unpublished data) Furthermore, mobile lipid signals have been reported in tumor cells exposed to lipophilic cationic compounds that cause mitochon-drial damage [57] Also the observed decrease of intra-cellular taurine in cells exposed to 2-PTS for 24 h (i.e before the occurrence of massive cell death at 48 h) seems to be in general agreement with the view of pro-gressive mitochondrial impairment in these cells In fact, Hansen et al [58] recently suggested a new role of taurine in mitochondrial function by acting as a modu-lator of pH in the mitochondrial matrix and therefore altering the overall oxidative capacity of this subcellu-lar organelle, including a reduction in fatty acyl b-oxi-dation According to this hypothesis, the simultaneous decrease in taurine and an increase in NMR-detected mobile lipids suggest a substantial 2-PTS-induced loss

of mitochondrial function with subsequent accumula-tion of long fatty acyl chains in triglycerides in cyto-plasmatic lipid bodies In recent years, it has become

A

B

Fig 7 Trx oxidation by 2-PTS, in the presence and in the absence

of rhodanese, by measurement of NADPH (50 l M ) consumption

(absorbance at 340 nm), in 50 m M Tris–HCl buffer, pH 8.0, 1 m M

EDTA, with 4 l M Trx, 0.1 U Trd and in the presence of 0.5 m M

2-PTS (A) Tris–HCl buffer (a) or 4 l M RhdA(ES) (b) was added to

the solution after 30 min at 37 C, and, after stabilization, 2-PTS

was added The data were normalized against an A 340 of 0.093

(which represents 100%) (B) Tris–HCl buffer (a) or 4 l M RhdA-PS

(b) was added to the solution after 15 min of incubation at 37 C

and, after stabilization (about 10 min), 2-PTS was added The data

were normalized against an A 340 of 0.085 (which represents

100%).

Trang 9

apparent that mitochondria are integrally involved in

the mechanism of cell death, and anticancer drugs,

which inhibit the functions of mitochondria can

sensi-tize the cells to undergo apoptosis [59,60] It is

impor-tant to consider that rhodanese is an imporimpor-tant

mitochondrial enzyme in mammalian cells [61] and,

certainly, the rhodanese–Trx system is involved in the

integrity of this main energy-generator organelle

It is noteworthy that recently it has also been

dem-onstrated that rhodanese deficiency affects the activity

of Fe–S enzymes of the tricarboxylic acid cycle, such

as the aconitase in A vinelandii [15] These studies

sug-gest that in tumor cells in which there is a decrease in

expression of the TST gene [19,20], the involvement of

rhodanese in the detoxification of OSCs could lead to

an inhibition of the normal function of the cyanide

detoxification and Fe–S repair activities of this

enzyme, thus inhibiting the normal functions of the

mitochondria The elucidation of interactions between

OSCs and proteins, such as rhodanese or Trx that are

involved in maintaining the redox homeostasis of the

cell, could help to explain the mechanism whereby they

can induce programmed cell death in tumor cells In

the present work we investigated the effect of 2-PTS

on the activity and expression of TST We performed

our experiments in vitro using the A vinelandii

rhoda-nese The data reported here demonstrate that 2-PTS

is able to interact with the Cys of RhdA(E), inducing

a covalent modified form (RhdA-PS) 2-PTS interacts

with the active site of the rhodanese enzyme by

thiola-tion of the catalytic cysteine, therefore inhibiting its

TST activity A peculiar characteristic of the A

vine-landiienzyme is the presence of only one cysteine

resi-due, which is the catalytic site The structural

similarity of RhdA with the bovine rhodanese led us

to speculate that 2-PTS can induce a thiolation at the

level of the catalytic Cys residue of the human

rhoda-nese The ability of this garlic compound to thiolate an

internal Cys, such as that of the active site of

rhoda-nese, is an important observation that should be borne

in mind when considering the mechanism of action of

OSCs We showed that the thiolation of a

mitochon-drial enzyme, which is involved in ‘managing’ the

redox state of the cell, could be a relevant event in the

mechanism of action of this compound

The major sensitivity of RhdA-PS to degradation by

proteolysis is an important factor that should be

con-sidered Limited proteolysis of RhdA-PS shows that,

as well the alkylation of bovine rhodanese [43], local

conformational changes occur when the enzyme is

modified by 2-PTS interactions, and high flexibility of

the enzyme with respect to RhdA(ES) is induced

These data were also in agreement with a significant

decrease in the expression of TST after 48 h of treat-ment with 2-PTS (data not shown) Therefore, the major flexibility of RhdA-PS may also explain its ability to interact with and oxidize the Trx compared

to RhdA(ES)

Our results highlight a direct interaction between OSCs from garlic and rhodanese The bond of the propenyl sulfide with the catalytic cysteine shows the characteristic of a disulfide bond (i.e it is not cleavable

by nucleophilic attack of the cyanide) The propenyl-sulfur protein has a low redox potential, so Trx and dithiothreitol reduce it to restore the TST activity These results provide evidence that, in vitro, reduced Trx also regulates TST activity via redox regulation 2-PTS alone was also able to oxidize the reduced Trx, but in the presence of RhdA(ES) or RhdA-PS Trx was oxidized more rapidly The results presented here sup-port a possible mechanism (Fig 8) where the reduced Trx could be the sulfide acceptor of the rhodanese in the intracellular system Trd was able to reduce the oxidized Trx, and thus this process may be a potential sulfane sulfur detoxification system present in the cell This result highlights the antioxidant action of a rhodanese– Trx–Trd system against OSCs As described in previous work, reduced Trx is a sulfur-acceptor substrate for rho-danese It has been also hypothesized that a primary function of the rhodanese could be Trx-linked oxygen radical detoxification [46,47] The data presented here shed new light on the role of this enzyme in the sulfane sulfur detoxification system and on the involvement of the Trx–Trd system in this process Recently, evidence for the down-regulation of TST expression in some cancer cells has been reported [20,21] Our data show that no change in the expression of rhodanese occurs after 8 and 24h of treatment with 2-PTS By contrast,

O

||

H 2 = H - H 2 - S - S - O - N a + - S _ R d

||

O

S T P -2

S _ S _

|

H 2

d R

H S

x T

S

-S _ S _ H 2 H = H 2

x

T S_ H -S _ R d

2

| H

||

H 2

H P D N d T

x

T S

S

| HS_ H 2 H = H 2

Trd

Fig 8 Scheme of the proposed reactions of the interaction between rhodanese (Rhd) and 2-PTS, and of the restoration of rho-danese activity by the thioredoxin (Trx)–thioredoxin reductase (Trd) system in the cell.

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a significant reduction of TST activity was observed

dur-ing treatment, indicatdur-ing that cyanide detoxification of

rhodanese was reduced by the presence of 2-PTS Thus,

the data suggest that rhodanese could be a target

enzyme of the garlic OSCs and that the reduced TST

activity could be caused by an increase of the

sulfur-detoxification activity of the enzyme, which also

involves the Trx system

The ability of 2-PTS to inhibit, either in vitro or in

the cell, the TST activity of the rhodanese and to

oxi-dize the Trx in vitro, both in the absence and in the

presence of rhodanese, can be related to its ability to

induce apoptosis in the cell Our studies showed that

the viability of HuT 78 cells is reduced significantly

after 24 h of exposure to 2-PTS and this reduced

growth rate is related to a blockage in the G2⁄ M phase

of the cell cycle HuT 78 cells underwent an early

increase of ROS flux after the addition of 2-PTS, and

2-PTS-treated cells showed a rapid and sustained

increase of GSH levels up to 12 h, most probably

because of a detoxification process These results

implied a strict correlation between the apoptotic

effects of 2-PTS and oxidative imbalance, and this can

also be linked to a reduction in the ability of the

rhoda-nese–Trx system to detoxify by oxygen radicals in vivo

Interestingly, the blockage in the G2⁄ M phase of the

cell cycle was linked to an early increase of ROS flux

Cells treated with 2-PTS showed a rapid and sustained

increase in GSH levels up to 12 h; this was attributable

to a detoxification process These results imply a strict

correlation between 2-PTS apoptotic effects and

oxida-tive unbalance, and this can be also linked to a

reduc-tion in the activity of the oxygen radicals detoxificareduc-tion

of the rhodanese–thioredoxin system

These results are in agreement with previous studies

reported by Chang et al [7], where it was observed

that 2-PTS suppresses, in a dose-dependent manner,

the growth of HL-60 cells through the induction of

apoptosis initiated by oxidative stress Although there

might be several mechanisms involved in the apoptosis

of cancer cells, we believe that the effects of this sulfur

compound, and probably also of other OSCs, may be

linked to mitochondrial expression levels and activity

both of rhodanese and of Trx in the cancer cells and

that the RNA interference technique could be used to

demonstrate this hypothesis

Interestingly, high levels of Trx have also been

associ-ated with cancer that is resistant to therapy, and low

Trx levels have been associated with apoptosis in gastric

carcinomas [62–64] The Trx⁄ Trd system is considered

to act as an endogenous antioxidant system in all living

cells, in addition to the glutathione system, so the

mal-function of this antioxidant system in mitochondria can

lead to an increase of intracellular ROS [65] Mitochon-drial rhodanese–Trx⁄ Trd-system oxidation by OSCs could lower the normal reducing activity of Trx and thus inhibit the activity of important enzymes, such as perox-iredoxin 3, an enzyme involved in H2O2 metabolism in mitochondria, whose oxidation plays an important role

in the promotion of apoptosis [66]

In conclusion, these studies contribute to extend the knowledge on the physiological role of rhodanese and may represent a relevant starting point to elucidate the implication of the rhodanese–Trx⁄ Trd system in che-moprevention therapy approaches using sulfane sulfur compounds, whose biochemical metabolism and certain biological effects warrant further investigation

Materials and methods

2-PTS synthesis Sodium 2-propenyl thiosulfate was synthesized according to

a method described by Chapelet et al [67] The product was dried in vacuo, extracted with methanol and the extract was purified by silica gel chromatography (methanol⁄ chloroform; 45 : 55, v⁄ v) The purity and structure of the compound were evaluated by RP-HPLC, LC-MS and

1H-NMR

Cell proliferation assay HuT 78 human T-lymphoblastoid cells were purchased from the ISS (Istituto Superiore di Sanita`, Rome, Italy) Approximately 0.2· 106

HuT 78 cells were pre-incubated for 24 h in RPMI 1640 (GIBCO, Milan, Italy) in the pres-ence of 1% glutamine, 10% heat-inactivated fetal bovine serum and antibiotics (1% penicillin and streptomycin sulfate) at 37C in air supplemented with 5% CO2 and were then exposed to 2-PTS for 24 and 48 h The cells were collected and counted, after staining with Trypan Blue (0.4% Tripan blu solution; Sigma-Aldrich, Milan, Italy), by optical microscopy using a Thoma chamber The rate of growth inhibition by 2-PTS was calculated with respect to the control culture taken as 100% growth

Cell cycle analysis The cell cycle distribution of HuT 78 cells was measured by flow cytometry Approximately 0.5· 106

harvested cells were stained with 50 lgÆmL)1 of propidium iodide (Sigma-Aldrich) in NaCl⁄ Pi with 0.1% Triton X-100 and

1 mgÆmL)1 of sodium citrate Then, the cells were imme-diately analysed using a flow cytometer (FACSCalibur; Becton Dickinson, San Jose`, CA, USA) and the percentage

of cells in each phase of the cell cycle was evaluated accord-ing to Nicoletti et al [68]

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