ELSEVIER

www.elsevier.nl/locate/jinorgbio

Journal of Inorganic Biochemistry 75 ( 1999) 255-261

IiW~anic Biochemistry

Synthesis and characterization of complexes of p-isopropyl benzaldehyde and methyl 2-pyridyl ketone thiosemicarbazones with Zn ( II) and Cd( II) metallic centers. Cytotoxic activity and induction

of apoptosis in Pam-ras cells

Jo& M. P&ez a,1, Ana I. Matesanz a,1, Alfonso Martin-Ambite a*2, Paloma Navarro a, Carlos Alonso b, Pilar Souza a,*

’ Departamento de Quimica Inorgcinica, Facultad de Ciencias, Universidad Autdnoma de Madrid, Cantoblanco, 28049 Madrid, Spain

h Centro de Biologia Molecular ‘Sever0 Ochoa’ (CSIC-UAM), Facultad de Ciencias, Universidad Autdnoma de Madrid, Cantoblanco, 28049 Madrid, Spain

Received 5 January 1999; received in revised form 1 June 1999: accepted 10 June 1999

Abstract

It is known that metallic complexes of methyl 2-pyridyl ketone thiosemicarbazone (HL’ ) andp-isopropyl benzaldehyde thiosemicarbazone (HL’) may have potential antitumor activity. We have prepared complexes of HL’ and HL* with Zn( II) and Cd( II). The cytotoxic activity shown by these compounds against cell lines sensitive and resistant to cis-diammincdichloroplatinum( II) (cis-DDP) indicates that coupling of HL’ and HL* to Zn(I1) and Cd(I1) centers may result in metallic complexes with important biological properties since they display It& values in a pM range similar to that of the antitumor drug cis-DDP. Moreover, it is interesting to note that the Zn/HL* complex exhibits specific cytotoxic activity against Pam-r-as cells (cis-DDP resistant cells which overexpress the H-ras oncogene) with an in vitro therapeutic index of 3.26 versus 0.78 for cis-DDP. Treatment of Pam-ras cells with the It& value of the Zn/HL’ compound induces a ‘DNA ladder’ (fragmentation of genomic DNA in nucleosome units) indicative of apoptosis in this rus-transformed cell line. In contrast, a ‘DNA smear’ (non-specific fragmentation of genomic DNA) is observed in Pam 212 normal cells treated with the IC5, of this compound. The analysis by circular dichroism (CD) spectroscopy of the interaction of the Zn/HL* compound with calf thymus DNA (CT DNA) indicates that it produces stronger alterations on the double helix conformation than cis-DDP. So, these results suggest that Zn/HL’ may be considered a potential antitumor agent. 0 1999 Elsevier Science Inc. All rights reserved.

Keywords: Cytotoxic activity; Apoptosis; Pam-ras cells; p-Isopropyl benzaldehyde thiosemicarbazones; Methyl 2-pyridyl ketone thiosemicarbazones

1. Introduction

Thiosemicarbazones, such as methyl 2-pyridyl ketone thio- semicarbazone (HL’) and p-isopropyl benzaldehyde tbio-

semicarbazone (HL’) have considerable pharmacological interest due to their antibacterial, antiviral and antitumor activities [ l-41. Moreover, it has been shown that the activity of the thiosemicarbazone compounds may be enhanced by the presence of some metallic ions due to their ability to form

coordination complexes [ 5,6]. Thiosemicarbazones usually react as ligands with metal

cations by bonding through the sulfur and the azomethinic

* Corresponding author. Tel.: 4 34-91-397-5 146; fax: + 34-91-397-4833;

e-mail: souza@jet.es

’ Both authors contributed equally to this work.

2 The paper is part of the directed study presented by this co-author in Environmental Science.

nitrogen atoms, although in some cases they behave as ter- dentate ligands bonded through the sulfur and two nitrogen

atoms [S-9]. The implications of zinc compounds in tumor growth inhibition have been the subject of several studies [ 10-121 and some zinc inorganic and complex compounds have been screened as antitumor agents but without obtaining promising results [7-9,12-141. However, antitumor prop- erties have been established for some cadmium and mercury compounds [ 15,161, although they are considered to be major poisons for living organisms.

It has been recently reported that several Pd( II) and Pt( II)

complexes with HL’ and HL* have potential antitumor activ- ity [l-6]. After this encouraging result we prepared other metallic complexes of HL’ and HL’ as a first step in studying whether metallic centers of Zn(II) and Cd(I1) enhance the pharmacological activity of the thiosemicarbazone ligand.

0162-0134/99/$ - see front matter 0 1999 Elsevier Science Inc. All rights reserved. PIISO162-0134(99)00096-3

2.56 J.M. Ptkez et al. /Journal of Inorganic Biochemistry 75 (1999) 255-261

M = Zn(ll), Cd(ll)

Fig. 1, Structures of the synthesized metal complexes derived from methyl

2-pyridyl ketone thiosemicarbazone andp-isopropyl benzaldehyde.

The results reported in this paper indicate that coupling of HL’ to Zn(I1) (1) and Cd(I1) (2), and HL’to Zn(I1) (3) and Cd( II) (4) may result in metallic complexes with impor- tant biological properties (Fig. I j since they exhibit cytotoxic activity in a pM range similar to that of the antitumor drug cis-DDP. Moreover, it is interesting to note that Zn/HL2 exhibits specific cytotoxic activity against cis-DDP resistant cells which overexpress the H-rus oncogene (Pam-rus cells). Treatment of Pam-ras cells with the IC,, of Zn/HL’ induces a ‘DNA ladder’ indicative of apoptosis. In contrast, a ‘DNA smear’ is observed in Pam 212 normal cells after treatment with this compound. These findings suggest that the specific cytotoxic activity shown by Zn/HL2 in Pam-ras is due to cell killing by apoptosis.

2. Experimental

2.1. Measurements

Elemental analyses (C, H, N and S) were carried out on Perkin-Elmer model 2400 automatic equipment. Infrared spectra (4000-400 cm- ’ j for KBr disks were recorded on a Bomen-Michelson spectrophotometer; electronic spectra, in DMF (dimethyl formamide) solution, were recorded on an Ati-Unicam UV2 spectophotometer. The circular dichroism (CD) spectra of CT DNA (calf thymus DNA) and CT DNA:drug complexes formed at r, =O.l (molar ratio of Pt(I1) either Zn(I1) or Cd( II) bound per nucleotide) were performed in a 1 cm rectangular quartz cell in a JASCO J- 600 spectropolarimeter attached to a temperature program- mer using a computer for spectral subtraction and noise reduction. The CD analysis was done at 37°C. Each sample was scanned twice in a range of wavelengths between 220 and 320 nm. The generated CD spectra represent the mean of three independent scans. The data are expressed as mean residue molecular elipticity ( (3) in degree cm2 dmoll ’ X 10’.

2.2. Materials

A general procedure was followed. To a methanolic solu- tion (25 ml) of HL2 (0.22 g, 1 mmol) at 50°C was added dropwise with stirring, a methanolic solution (25 ml j of metal chloride (Zn or Cd) (0.5 mmol) . After 2 h of contin- uous stirring was added dropwise 10 ml of HC1(35%). The solid was isolated after slow evaporation of the solvent, washed with MeOH and Et,0 and dried in vacua.

Thiosemicarbazide, methyl 2-pyridyl ketone, p-isopropyl [Zn(HL2jCl,] (3): yield 45%. Found: C, 35.69; H, 3.43; benzaldehyde, ZnCl, and CdCl, were commercially available N, 11.07; S, 8.67. C,,H,,Cl,N,SZn, MW 285.55, requires: and used without further purification. C, 36.90; H, 4.20; N, 11.75; S, 8.95%.

2.2.1. Biological reagents and drugs The 100 mm culture and microwell plates were obtained

from Nunclon (Roskilde, Denmark) ; 3- [ 4,5dimethylthia- zol-2-yl] -2,5-diphenyltetrazolium bromide (MTT) was pur- chased from Sigma; fetal calf serum (FCS) was supplied by GIBCO-BRL; cis-diamminedichloroplatinum( II) (cis- DDP) was purchased from Sigma. Metal complexes and cis- DDP were dissolved in 10 mM NaClO,. Stock solutions of the compounds at a concentration of 1 mg/ml were freshly prepared before use.

2.2.2. Cell lines and culture conditions HeLa (cervix epithelial human carcinoma line) and Vero

(transformed monkey kidney fibroblasts) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supple- mented with 10% fetal calf serum (FCS) and 10% of new born calf serum (NBCS), respectively, together with 2 mM glutamine, 100 units/ml penicillin, and 100 mg/ml strepto- mycin at 37°C in an atmosphere of 95% of air and 5% CO,. Pam 212 (normal murine keratinocytes) and Pam-ras (murine keratinocytes transformed with the H-r-us oncogene and resistant to cis-DDP) [ 17,181 were cultured in DMEM supplemented with 10% FCS and 2 mM glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin at 37°C in an atmosphere of 95% of air and 5% COz.

2.3. Preparations

Methyl 2-pyridyl ketone thiosemicarbazone (HL’ j and p- isopropyl benzaldehyde thiosemicarbazone ( HL2) were pre- pared using published procedures [ 19,201.

2.3.1. [Zn(HL’)Cl,] (I), [Cd(HL’)ClJ (2) A general procedure was followed. To a methanolic solu-

tion (25 ml) of HL’ (0.20 g, 1 mmol) at 50°C was added dropwise with stirring a methanolic solution (25 ml) of metal chloride (Zn and Cd) (0.5 mmol) After 1 h of continuous stirring the solid was isolated, washed with methanol (MeOH) and ether (Et,O) and dried in vacua.

[Zn(HL’jCl,] (1): yield 28%. Found: C, 29.65; H, 3.15; N, 16.40; S, 9.65. C,H,,Cl,N,SZn, MW 330.40, requires: C, 29.05; H, 3.00; N, 16.95; S, 9.70%.

[ Cd( HL’ )Cl,] (2) : yield 38%. Found: C, 26.00; H, 2.70; N, 15.00; S, 8.70. CsH,&dC12N$, MW 377.45, requires: C, 25.40; H, 2.65; N, 14.85; S, 8.45%.

2.3.2. [Zn(HL2)C12] (3), [Cd(HL’)Cl,]. 2H,O (4)

J.M. Ptrez et al. /Journal of Inorganic Biochemistry 75 (1999) 255-261 251

[ Cd( HL*) Clz] . 2H20 (4) : yield 53%. Found: C, 28.50; H, 4.40; N, 7.65; S, 6.52. C,,H&dC12N302S, MW 440.40, requires: C, 29.90; H, 4.30; N, 9.50; S, 7.20%.

2.4. Drugs cytotoxicity

Cell proliferation was evaluated by using a system based on the tetrazolium compound MTT which is reduced by living cells to yield a soluble formazan product that can be assayed calorimetrically [ 211. Cells were plated in 96-well sterile plates, at a density of lo4 cells/well in 100 ~1 of medium, and were incubated for 34 h. Stock solutions of the com- pounds dissolved in DMEM were added to the wells at final concentrations from 0 to 200 p,M, in a volume of 100 kl/ well. After 24 or 96 h of incubation, 50 ~1 of a freshly diluted MTT solution ( l/5 in culture medium) was added to a final concentration of 1 mg/ml into each well and the plate was further incubated for 5 h. Cell survival was evaluated by measuring the absorbance at 520 nm, using a Whittaker 2001 microplate reader. IC50 values were calculated from curves constructed by plotting cell survival (%) versus compound concentration (FM). All experiments were made in quadruplicate.

roform/isoamylalcohol (24: 1) phase. Subsequently, the DNA was precipitated overnight at -20°C in 2.5 volumes of cold 100% ethanol/ 150 mM potassium acetate. After cen- trifugation at 12 000 rpm for 15 min to recover the precipi- tated DNA the supernatant was discarded and the pellet was washed with 70% ethanol. Samples were dried using a SAVANT speed vat concentrator and then resuspended in distilled water. The DNA concentration was calculated by determining the OD26,,. Electrophoresis of DNA (10 Fg/ well) was performed for 16 h at 75 V in 1.8% agarose gel with TAE (40 mM Tris-Acetate, 1 mM EDTA, pH 8.0) as running buffer. DNA was visualized by ethidium bromide staining for 16 h and UV trans-illumination. DNA bands were analyzed by laser densitometry using a Molecular Dynamics densitometer.

2.6. Formation of compound:DNA complexes

2.5. DNA fragmentation assay

Pam 212 and Pam-ras cells (5 X lo5 cells/ml) were plated in 100 mm sterile dishes. The cells were treated with the I&, of the compounds for 1, 10, 24 and 72 h under the above- mentioned conditions. The fraction of detached cells was collected by centrifugation of the culture media and washed twice with phosphate-buffered saline (PBS). The cell pellet was disrupted with 700 ~1 of lysis buffer (150 mM Tris, tris(hydroxymethyl)aminomethane, pH 8.0; 100 mM NaCl; 100 mM EDTA, ethylenediaminetetracetate) . The fraction of non-detached cells was also washed twice with PBS and lysed by addition to the plate of 700 ~1 of lysis buffer. Both cell fractions were joined and the whole cell lysate was treated with proteinase K (500 pg/ml) for 2 h at 55°C. Afterwards, samples were exposed to RNase A (50 p,g/ml) for 16 h at 37°C. The DNA was first extracted with phenol, followed by phenol/chloroform/isoamylalcohol (25:24: 1) and a chlo-

The formation of the compound:DNA complexes was done by incubation in 10 mM NaClO, at 37°C of CT DNA with a given volume of stock solutions of the synthesized com- pounds or cis-DDP until r,, = 0.1 is achieved (molar ratio of Zn(II), Cd(I1) or Pt(II) bound to nucleotides). The unreacted compound molecules were separated from the mix- ture by precipitation of the DNA with 2.5 volumes of ethanol and 0.3 M NaAc, pH 4.8. Platinum, zinc or cadmium atoms bound to CT DNA were quantified by total reflection X-ray fluorescence (TXRF) [ 22,23 1.

3. Results and discussion

3.1. Infrared and UV-Vis spectra

The main vibrational bands of free ligands and their com- plexes are compared in Table 1. The assignment of the bands in the 3500-3100 cm- ’ region is difficult owing to the pres- ence of NH groups and numerous hydrogen-bonding inter- actions (NH...N and NH...S). A very strong peak at 1589 cm -’ in HL2 has been assigned to the superimposed modes u( GN) azomethine and 6(NH,). The spectrum of HL’

Table 1

Selected vibrational bands (cm-‘) and solution (DMF) electronic spectra (nm) of free ligands and their metal complexes

HL’ 1 2 HL’ 3 4

dNH) 3363,3257,3 180 3267,3 195,3086 3302,3295,3116

NNH,) 1608 1615 1612

v( C=N) 1582 1610 1602

v(C=N),,+ v(C=C) 1560 1598 1568

Thioamide 1 1500 1557 1554

Thioamide II 1426 1443 1441

Thioamide III 1149 1170 1168

Thioamide IV 849 803 799

Intraligand band 320 310 315

Charge transfer band - 395 385

3276,3 148

1589

1589

1570

1529

1420

1185

873

325

3266,3159

1597

1574

1534

1440

1187

838

320

360

3271,3177

1631

1607

1576

1560

1422

1189

837

320

380

258 J&l. P6rez et al. /Journal of Inorganic Biochemistry 75 (1999) 2X-261

shows two peaks at 1582 and 1608 cm-’ assigned to v( C=N) and S( NH,), respectively. The positive shift of the v(C=N) band in the complexes illustrates participation of the imine nitrogen atom in the coordination. Also, all the complexes show significant changes in the 1550-700 cm-’ region (thioamide I-IV bands) ; the negative shift in the thioamide IV band is consistent with coordination through the sulfur atom of the thioamide group [ 241. Therefore, these data indicate that the ligands act in a bidentate form through the imino and thioamide groups in these complexes.

Also included in Table 1 are the band maxima of solution (dimethylformamide) electronic transitions for HL’, HL’ and their complexes. The n + n* transitions at about 320 and 325 nm, for HL’ and HL* respectively, primarily associated with the azomethine functions of the thiosemicarbazone moi- eties, are shifted to higher energy for the complexes. In addi- tion, the metal complexes have a ligand-to-metal charge transfer transition (from S +M and N+M) which is dis- placed to higher energies from Zn to Cd HL’ complexes and to lower energies from Zn to Cd HL* complexes [ 251.

3.2. Cytotoxic activity of the thiosemicarbazone-metallated compounds

We have tested the cytotoxic activity of the synthesized compounds and the antitumor drug cis-DDP against tumor cell lines sensitive to cis-DDP ( HeLa, Vero), resistant to cis- DDP (Pam-ras), and normal cells (Pam 2 12) after treatment periodsof24h(Table2(A))and96h(Table2(B)).Itmay be observed in Table 2(A) that the metallic thiosemicarba- zone complexes have I& in a pM range similar to that of cis-DDP. Table 2(A) also shows that Cd/HL* and Zn/HL* are 43 times and about 3 times, respectively, more active than cis-DDP in the cis-DDP-resistant cell line Pam-ras (IC& values of 2.9 and 45 pM versus 124 PM, respectively). Moreover, Zn/HL* has a better in vitro therapeutic index (T.I. = IQ,, of parental cell line/K,, of transformed cell line) relative to Cd/HL’ and cis-DDP (T.I. of 3.26 versus 1.03 and 0.78, respectively). Similar data were obtained for a drug-treatment period of 96 h. However, the cytotoxic activ- ity observed was on average 3 to 4 times lower than that obtained at 24 h (Table 2(B) ) . Moreover, the data of Table 2 show that the HL* and HL’ ligands exhibit poor cytotoxic activity in all the cell lines tested. Altogether, these results indicate that in Zn/HL’ the covalent binding of the HL* ligand to a Zn( II) center results in a Zn-thiosemicarbazone compound with remarkable cytotoxic activity. Thus, Zn/HL* might have interesting antitumor properties, particularly in view of the fact that it is capable of circumventing cis-DDP resistance in Pam-ras cells.

3.3. Induction of apoptosis in Pam-ras cells by the metallated-thiosemicarbazone compounds

It has been recently reported that the ability of certain anticancer drugs to achieve a significant therapeutic index

Table 2

It& mean values and standard deviations (SDS) obtained for the synthesized

compounds and cis-DDP against several tumor and normal cell lines at 24

h (A) and 96 h (B) drug-treatment periods

Cell line Vero Pam 212 Pas-ms HeLa

(A) Zn/HL’

Cd/HL’

Zn/HL’

Cd/HL*

cis-DDP

HL*

HL’

(B) Zn/HL’

Cd/HL’

Zn/HL’

Cd/HL’

cis-DDP

HL*

HL’

53+4 37+2 84+3 41+2

25+2 3.0+0.7 2.9 k 0.5 lo*1

131*3 147+6 45*1 57+4

133&X5 60&4 93*5 45&2

164&4 97+3 124+6 40+3

> 200 >200 >200 >200

>200 >200 > 200 > 200

12+2 10+2 28&4 14*1

9+2 1.0*0.2 0.7&0.1 4*2

31+_5 36+3 10+1 16+_2

43+4 25k2 33+4 15+3

41*3 20+1 43*5 17*2

>lOO > 100 >ltXl >lOO

>lOO > 100 >lOo >lOO

differentiating normal cells from malignant cells may be related to the fact that they induce apoptosis in tumor cells at drug concentrations significantly lower than those needed to kill normal cells [ 26,271. In order to know whether Zn/HL*, i.e., the compound that showed a better in vitro therapeutic index, produces cell killing by apoptosis we carried out DNA laddering assays in Pam-ras and Pam 2 12 cells.

The results of the gel electrophoresis of DNA extracted from control Pam-ras cells and Pam-ras cells treated for 24 h with the IC,, of Zn/HL*, cis-DDP and the HL* ligand are shown in Fig. 2. Just as expected, it was observed that the DNA of control Pam-ras cells appears as a band correspond- ing to genomic DNA (Fig. 2, lane 1) . Incubation of the Pam- ras cells with the IC,, of cis-DDP or the HL’ ligand resulted in a ‘DNA smear’ (Fig. 2, lanes 2 and 3, respectively). Interestingly, however, a ‘DNA ladder’ indicative of apop- tosis was obtained in Pam-ras cells treated with the IC,, of Zn/HL* (Fig. 2, lane 4). The results of the gel electrophoresis

kb

- 4.37

- 2.87

- 0.76

Fig. 2. Agarose gel electrophoresis of DNA extracted from control Pam-ras

cells (lane 1) and Pam-ras cells treated with the IC,, of cis-DDP (lane 2))

HL’ ligand (lane 3), and Zn/HL* compound (lane 4). Lane 5: Hind III

digested 429 phage DNA.

J.M. Pkez et al. /Journal of Inorganic Biochemistry 75 (1999) 255-261 259

0.76 - Fig. 3. Agarose gel electrophoresis of DNA extracted from control Pam 212

cells (lane 2) and Pam 212 cells treated with the IC,, of HL* ligand (lane

3), Zn/HL’ compound (lane 4), and cis-DDP (lane 5). Lane 1: Hind III

digested $29 DNA.

of the DNA extracted from control Pam 212 cells and Pam 212 cells treated for 24 h with the I& of Zn/HL*, cis-DDP,

and the HL* ligand are shown in Fig. 3. It may be observed that, in contrast to that observed in Pam-rus cells, all the

compounds produce a smear of DNA (lanes 3,4 and 5). Fig. 4 shows the percentage of DNA laddering as a function

of the period of incubation with Zn/HL2, cis-DDP and the HL2 ligand obtained from densitometric analysis of the gels.

It was observed that, after 3 h of incubation with the IQ,, of Zn/HL2, 30% of DNA laddering is produced with 75% of

DNA laddering after 24-28 h. The percentage of DNA lad- dering decreased after 28 h of treatment; the value of 7% after

96 h of incubation is an indication that most of the cells died as a consequence of apoptosis. Interestingly, neither cis-DDP

nor HL* induces DNA laddering at any of the drug-treatment

periods tested.

Fig.

60 +

4.

20 I

T lo T

Altogether, these data suggest that in Pam-ras cells the higher cytotoxic activity of Zn/HL’ relative to the HL2 ligand and cis-DDP may be due to a specific induction of apoptosis.

3.4. Analysis of the interaction of ZrdHL’ with calfthymus

DNA (CT DNA)

Since Zn/HL2 is active in cis-DDP resistant cells and DNA is the main target of metal-based drugs [ 281, we have ana- lyzed the effect of binding of Zn/HL2 on CT DNA by means of CD spectroscopy. The CD spectra and the wavelengths at which the maximum and minimum values of elipticity ( 0) occur in control CT DNA and in CT DNA incubated with Zn/HL* and cis-DDP at r,= 0.1 are shown in Fig. 5 and Table 3, respectively. It may be observed that the maximum value of elipticity of the positive band at 276 nm in native CT DNA increases from 403 8 units to 440 8 units in Zn/ HL2:CT DNA complex. This change in elipticity is accom- panied by a small change to shorter wavelength of the maximum of the positive band which is located at 275 nm in

Zn/HL’:CT DNA complex. In cis-DDP:CT DNA complex the maximum value of elipticity of the positive band is located at 277 nm and decreases to 331 0 units. On the other hand, the minimum value of elipticity of the negative band present in native CT DNA at 249 nm decreases from - 382 0 units to - 379 0 units in Zn/HL*:CT DNA complex and is located at 248 nm as in control CT DNA. cis-DDP also induces a decrease in the minimum of elipticity of the negative band ( - 3 14 8 units) which is located at 249 nm as in control CT DNA. These changes produced in the CD spectrum of CT DNA by Zn/HL* are indicative of an opening and rotation of the stacked bases on the DNA double helix since the

Pam-ras cells

y , . , , , , , , . , , . . . , , , , , , , . . ,_ , , . , , . , , . . t..> , , . . . . . , I , . . , . , .Y,, . . , _ ,....^> . , . . . . . , , , . , . , . , . . . . , . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I . ,_ . . . . . . . . . . , . . . . . . . I . . . . . . . . . I I . . . . . . . . . . .1. . . . . . . . . . . .

0 hours 0 hours 3 hours 3 hours IO hours IO hours 24 hours 24 hours 28 hours 28 hours 46 hours 46 hours 72 hours 72 hours 96 hours 96 hours

time (hours) time (hours)

Percentage of DNA fragmentation in Pam-ras cells vs. incubation time at the IC,, Percentage of DNA fragmentation in Pam-ras cells vs. incubation time at the IC,, of Zn/HL* compound, cis-DDP and HL’ ligand. of Zn/HL* compound, cis-DDP and HL’ ligand.

260 J.M. Pkrez et al. /Journal of Inorganic Biochemistry 75 (1999) 255-261

5

4

2

“0

2 : z E

-a

“E 0

. 2 0 -0

z

-2

-4 220 240 260 280 300 J)Ld

Wavelength (nm)

Fig. 5. CD spectra of control CT DNA (- - -) and of CT DNA incubated with Zn/HL* (- ) and with cis-DDP (- - -) at rh = 0. I.

Table 3

CD spectral data a for Zn/HL’:CT DNA and cis-DDP:CT DNA complexes

formed at r, = 0.1

Complex

Native CT DNA 403 276 - 382 249

Zn/HL?CT DNA 440 275 - 319 248

cis-DDP:CT DNA 331 277 -314 249

’ 0 in degree cm* dmol- ’ X IO?, mean residue molecular elipticity; A in nm.

conservative nature of the CD spectrum is not altered and, in addition, there is a slight displacement of the CD curve towards longer wavelength [ 291. The differences observed in the CD spectrum of Zn/HL*:CT DNA complex relative to cis-DDP:CT DNA complex indicate that Zn/HL2 produces

stronger conformational changes on DNA secondary struc- ture than cis-DDP.

Taking into account that Zn/HL’ is active in cis-DDP resistant Pam-rus cells and, moreover, it induces more drastic conformational changes on DNA secondary structure than those induced by cis-DDP, it is likely that this Zn( II)-thio- semicarbazone compound might have potential antitumor

properties.

Acknowledgements

We thank the Comision Interministerial de Ciencia y Tec- nologia (Spain) for financial support (Project Nos. MAT 95

0934-E, SAF-96-0041 and BIO-096-040.5) and Dr Ramon y

Cajal from Clinica ‘Puerta de Hierro’ of Madrid for his kind

gift of Pam 212 and Pam-ras cells. An institutional grant from Fundacion Ramon Areces is also acknowledged.

References

[I] D.X. West, A.E. Liberta, S.B. Padhye, R.C. Chikate, P.B. Sonawane,

A.S. Kumbhar, R.G. Yerande, Coord. Chem. Rev. 123 (1993) 49.

[2] T.W. Hambley, Coord. Chem. Rev. 166 (1997) 181.

[3] C. Shipman Jr., S.H. Smith, J.C. Drach, D.L. Klayman, AntiviralRes.

6 (1986) 197.

[4] S.N. Pandeya, J.R. Dimmock, Pharmazie 48 ( 1993) 659.

[5] P. Souza, A.I. Matesanz, V. Fernindez, J. Chem. Sot., Dalton Trans.

(1996) 3011.

[6] A.G. Quiroga, J.M. Perez, I. Lopez-Solera, J.R. Masaguer, A. Luque,

P. Roman, A. Edwards, C. Alonso, C. Navarro-Ranninger. J. Med.

Chem. 41 (1998) 1399.

J.M. P&ez et al. /Journal of Inorganic Biochemistry 75 (1999) 255-261 261

[ 71 D.H Petering, H.G. Petering, in: A.C. Sartorelli, D.G. Johns (Eds.),

Handbook of Experimental Pharmacology, Springer, Berlin, 1975, p.

841.

[ 81 D.H Petering, W.E. Antholine, L.A. Saryan, in: R.M. Ottenbrite, C.B.

Butler (Eds.), Anticancer and Interferon Agents, Synthesis and Prop-

erties, Marcel Dekker, New York, 1984, p. 203.

[9] P. Souza, L. Sanz, V. Femindez, A. Arquero, E. Gutierrez, A. Monge,

2. Naturforsch., Teil B 46 ( 1991) 767.

[lo] M.M. Jacobs, A.C. Griffin, in: M.S. Zedeck, M. Lipkin (Eds.), Inhi-

bition of Tumor Induction and Developments, Plenum, New York,

1981, p. 169.

[ 1 l] A.M. van Rij, W.J. Pories, in: H. Sigel (Ed.), Metal Ions in Biological

Systems, vol. 10, Marcel Dekker, New York, 1980, p. 7.

[12] D.H. Barth, P.M. Iannaccone, Adv. Exp. Med. Biol. 206 ( 1986)

517.

[ 131 G.J. van Giessen, J.A. Crim, D.H. Petering, H.G. Petering, J. Natl.

Cancer Inst. 5 1 ( 1973) 139.

[ 141 G. Atassi, P. Dumont, J.C.E. Harteel, Eur. J. Cancer 15 ( 1979) 451.

[ 151 D. Solaiman, L.A. Saryan, D.H. Petering, J. Inorg. B&hem. 10

(1979) 135.

[ 161 G.C. Skvortsova, A.I. Skushnikova, E.S. Dommina.1.G. Veksler,K.P.

Balitskii, M.G. Voronkov, Khim.-Farm. Zh. 18 ( 1984) 679.

[ 171 M. Florin-Kristensen, C. Missero, J. Florin, P. Tranque, S. Ramon y

Cajal, G.P. Dotto, Exp. Cell. Res. 207 (1993) 57.

[ 181 R. S&rchez-Prieto, J.A. Vargas, A. Carnero, E. Marchetti, J. Romero,

A. Durantez, J.C. Lacal, S. Ramon y Cajal, Int. J. Cancer 60 (1995)

235.

[ 191 AI. Vogel, Practical Organic Chemistry, 4th ed., Longman, London,

1978, p. 1112.

[ 20 ] P.P.T. Sah, T.C. Daniels, Reel. Trav. Chim. Pays-Bas69 ( 1950) 1545.

(211 M.C. Alley, D.A. Scudiero, A. Monks, M.L. Huresey, M.J.

Czerwinski, D.L. Fine, B.J. Abbott, J.G.May0,R.H. Shoemaker,M.R.

Boyd, Cancer Res. 48 (1988) 589.

[ 221 P. Wobrauscheck, Biol. Trace Elem. Res. 43-45 ( 1994) 65-7 1.

[ 231 V.M. Gonzalez, P. Amo-Ochoa, J.M. Perez, M.A. Fuertes, J.R.

Masaguer, C. Navarro-Ranninger, C. Alonso, J. Inorg. Biochem. 63

(1996) 57.

[ 241 K. Nakamoto, Infrared and Raman Spectra of Inorganic and Coordi-

nation Compounds. Part B: Applications in Coordination, Organo-

metallic and Bioinorganic Chemistry, 5th ed., Wiley-Interscience,

New York, 1997.

[ 251 A.B.P. Lever, Inorganic Electronic Spectroscopy, 2nd ed., Elsevier,

Amsterdam, 1984.

[26] S.W. Lowe, H.E. Ruley, T. Jacks, D.E. Housman, Cell 74 ( 1993) 957.

[27] N.A. Jones, J. Turner, A.J. McIlwrath, R. Brown, C. Dive, Mol. Phar-

macol. 53 (1998) 819.

[ 281 I. Haiduc, C. Silvestru, Coord. Chem. Rev. 99 ( 1990) 253.

[29] W.C. Johnson, M.C. Itzkowic, I. Tinoco, Biopolymers 11 ( 1972) 225.