A cycloplatinated compound of p-isopropylbenzaldehyde thiosemicarbazone and its chloro-bridged...

0162-0134/99/$ - see front matter q 1999 Elsevier Science Inc. All rights reserved.PII S0162- 0134 (99 )00021 -5

Friday Apr 30 11:56 AM StyleTag -- Journal: JIB (Journal of Inorganic Biochemistry) Article: 6158

Journal of Inorganic Biochemistry 73 (1999) 235–243

A cycloplatinated compound of p-isopropylbenzaldehydethiosemicarbazone and its chloro-bridged derivative induce apoptosis

in cis-DDP resistant cells which overexpress the H-ras oncogene

Jose M. Perez a, Adoracion G. Quiroga a, Eva I. Montero a, C. Alonso b, C. Navarro-Ranninger a,*´ ´a Departamento de Quımica Inorganica, Facultad de Ciencias, Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid, Spain´ ´ ´

b Centro de Biologıa Molecular ‘Severo Ochoa’ (CSIC-UAM), Facultad de Ciencias, Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid, Spain´ ´

Received 14 September 1998; received in revised form 26 January 1999; accepted 5 February 1999

Abstract

cis-Diamminedichloroplatinum(II) (cis-DDP) is a widely used antitumour drug which produces important damage on the DNA inducingapoptosis in several cell lines. We have analysed the cytotoxic activity of novel cyclometallated complexes of p-isopropylbenzaldehydethiosemicarbazone (p-is.TSCN) and their dimeric chloro-bridged derivatives in murine keratinocytes transformed by the H-ras oncogenewhich are resistant to cis-DDP (Pam-ras cells). The data show that, in contrast with cis-DDP, the tetrameric cycloplatinated complex [Pt(p-is.TSCN)]4 and its dimeric chloro-bridged derivative [Pt(mCl)(p-is.TSCN)]2 have a good in vitro therapeutic index when comparing thecytotoxicity in Pam-ras cells to normal murine keratinocytes (Pam 212 cells) since they induce cell death in Pam-ras cells at drugconcentrations significantly lower than those needed to kill Pam 212 cells. At equitoxic doses (IC90), both complexes produce characteristicfeatures of apoptosis in Pam-ras cells together with a drastic decrease in levels of H-ras protein. These effects are not observed when the cellsare treated with the IC90 of the cis-DDP drug nor the p-is.TSCN ligand. Altogether, these results suggest that the platinum compounds [Pt(p-is.TSCN)]4 and [Pt(mCl)(p-is.TSCN)]2 might have potential as antitumour agents in view of their specific induction of apoptosis in cis-DDP resistant cells. q 1999 Elsevier Science Inc. All rights reserved.

Keywords: cis-DDP; Cyclometallated compounds; Cytotoxicity; Apoptosis; H-ras

1. Introduction

Apoptosis is a physiological mechanism for the eliminationof cells which occurs during embryonic development, hor-mone-induced atrophy and normal cellular homeostasis. Ithas been observed that apoptosis is also involved in particularcases of drug-induced tumour cell death [1–5]. Morpholog-ical changes associated with apoptosis include chromatincondensation and fragmentation of the nucleus [6] and pack-aging of cell remnants into ‘apoptotic bodies’, which are thenrapidly removed by macrophages [7]. One biochemicalmarker of apoptosis in many cell types is the cleavage ofDNA into 180-base pair integer fragments by a specificendonuclease [8].

Many currently used anticancer drugs kill particular typesof tumour cells by means of apoptosis [9,10]. Thus, cis-diamminedichloroplatinum(II) (cis-DDP) is a widely usedanticancer agent that induces apoptosis in various types of

* Corresponding author. Tel.: q34-91-397-4356; fax: q34-91-397-4833;e-mail: [email protected]

tumour cell lines such as HL-60, Chinese hamster ovary, andJB1 hepatoma cells [11–13]. cis-DDP causes the formationof several types of DNA lesions, mainly, intra- and interstrandDNA crosslinks [14–16] and it has been proposed that theamount of apoptosis after exposure to cis-DDP depends onthe levels of unrepaired DNA crosslinks [17]. Since apop-totic cells are processed by macrophages while necrotic cellslysate and release their constituents to the extracellularmedium producing local toxicity, we think that the search forplatinum derivatives with cytotoxic activity in cis-DDP resis-tant tumour cells, due to apoptosis, may lead to less toxicdrugs for the chemotherapy of cancer.

Recently, we have reported the synthesis, structural char-acterization and cytotoxic activity of cyclometallatedcomplexes of p-isopropylbenzaldehyde thiosemicarbazone(p-is.TSCN) and of their dimeric chloro-bridged derivativesshowing that these compounds are active in cis-DDP resistantcell lines [18,19].

In the present paper we have analysed the cytotoxicityinduced by these novel metallated complexes of p-isopro-

J.M. Perez et al. / Journal of Inorganic Biochemistry 73 (1999) 235–243´236


pylbenzaldehyde thiosemicarbazone in cis-DDP resistantcells that overexpress the H-ras oncogene (Pam-ras cells).The data show that, in contrast with cis-DDP, the tetramericcycloplatinated compound [Pt(p-is.TSCN)]4 and its dimericchloro-bridged derivative [Pt(mCl)(p-is.TSCN)]2 have agood in vitro therapeutic index since they induce cell deathin Pam-ras cells at drug concentrations significantly lowerthan those needed to produce a similar effect in normalmurinekeratinocytes (IC90 values of 40 versus 85 mM and of 50versus 80 mM, respectively). Moreover, at the IC90, thesecomplexes induce characteristic properties of apoptosis inPam-ras cells such as ‘DNA laddering’ and fragmentednuclei together with a drastic decrease in H-ras protein levels.Interestingly, however, these two effects are not produced bythe cis-DDP drug, nor the p-isopropylbenzaldehyde thio-semicarbazone ligand.

2. Materials and methods

2.1. Reagents and drugs

100 mm (P100) and 60 mm (P60) culture and microwellplates were obtained from NUNCLON (Roskilde, Den-mark). 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazo-lium bromide (MTT) was purchased from Sigma, foetal calfserum (FCS) was supplied by GIBCO-BRL, and ethanol wasobtained from Merck. cis-DDP (cisplatin), Mws300, wassynthesized by the method of Dhara from K2PtCl4 suppliedby Johnson-Mathey Co. [20]. Etoposide (podophilotoxin),Mws588.58, was obtained from Sigma. Adriamycin (doxo-rubicin), Mws543.54, was purchased from TEDEJI-Farmaand taxol (paclitaxel), Mws853.92, was supplied by Bristol-Myers Squibb Co. The thiosemicarbazone compounds p-is.TSCN, 1, Mws221; [Pd(mCl)(p-is.TSCN)]2, 2,Mws724.37; [Pt(mCl)(p-is.TSCN)]2, 3, Mws901.69;[Pd(p-is.TSCN)]4, 4, Mws1302.90; and [Pt(p-is.TSCN)]4, 5, Mws1657.54, were synthesized as describedpreviously [18,19].

cis-DDP and adriamycin were dissolved in phosphate buf-fer saline (PBS) as 5 mg/ml stock solutions. Etoposide wasdissolved in ethanol as 1 mg/ml stock solution and taxol wasdissolved in 50% dehydrated ethanol/50% castor oil as 6mg/ml stock solution. The thiosemicarbazone complexeswere dissolved in 50% ethanol/50% PBS as 5 mg/ml solu-tions. These solutions were freshly prepared before use.

2.2. Cell lines and culture conditions

Pam 212, a normal murine keratinocytes cell line, andPam-ras, a transformed murine keratinocytes cell line resistant tocis-DDP that overexpresses the H-ras-oncogene [21], weregrown in Dulbeco Modified Eagle’s Medium (DMEM) sup-plemented with 10% foetal calf serum (FCS), 2 mM gluta-mine, 100 units/ml penicillin, and 100 mg/ml streptomycinat 378C in an atmosphere of 95% air and 5% CO2. The main

mechanism of resistance to cis-DDP of Pam-ras cells is dueto an increase in the intracellular levels of glutathione so thata large amount of SH groups are able to coordinate to cis-Ptmolecules [22]. Pam 212 cells were passaged twice weeklyshowing a doubling time of 48 h and Pam-ras cells werepassaged three times weekly showing a doubling time of24 h.

2.3. Cytotoxicity assays

Survival of Pam 212 and Pam-ras cells was evaluated byusing a system based on the tetrazolium compound MTT,which is reduced by living cells to yield a soluble formazanproduct that can be detected colorimetrically [23]. Cellswereplated in 96-well sterile plates at a density of 104 cells/wellin 100 ml of medium and were incubated for 3–4 h. Thecompounds dissolved in DMEM medium containing 10% ofethanol were added to the wells at final concentrations from0 to 250 mM in a volume of 100 ml to achieve a final 5%ethanol concentration. After 24 and 96 h, 50 ml of a freshlydiluted MTT solution (1/5 in culture medium) to a concen-tration of 1 mg/ml were pipetted into each well and the platewas incubated for 5 h at 378C in a humidified 5% CO2 atmos-phere. After the specified periods, the cell viability was eval-uated by measurement of the absorbance at 520 nm, using aWhittaker Microplate Reader 2001. IC90 values (compoundconcentration that produces 10% of cell survival) were cal-culated from curves constructed by plotting (%) cell survivalversus drug concentration. The therapeutic index (T.I.) ofevery compound was calculated as the ratio of the IC90 innormal Pam 212 cells versus the IC90 in transformed Pam-ras cells. In control experiments it was observed that a 5%concentration of ethanol had no effect on cell survival or cellmorphology. The stability and speciation of the compoundsin culture medium containing ethanol in several proportions(0.1, 1 and 5%) were evaluated by purifying the chemicalspecies by HPLC and further analysis by NMR spectroscopyand potentiometric studies. It was observed that, even at 5%ethanol in culture medium, the thiosemicarbazone com-pounds were stable up to 96 h forming active species. Allexperiments were made in quadruplicate.

2.4. Changes in cell morphology

For the examination of the cell morphology associatedwithapoptosis, control and treated cells were photographed byphase contrast using a Zeiss Axiovert 35 microscope(=200). The cells were plated in P100 plates at a density of5=105 cells/ml. Afterwards, the drugs were added to theplates at equitoxic concentrations (IC90) and incubated withthe cells for 10 h.

2.5. DNA fragmentation assay

Cells (5=105 cells/ml) were plated in 100 mm steriledishes. The cells were treated with the IC90 of the compounds

J.M. Perez et al. / Journal of Inorganic Biochemistry 73 (1999) 235–243´ 237


for 24 h under the above-mentioned conditions. The fractionof detached cells was collected by centrifugation of the cul-ture media and washed twice with PBS. The cell pellet wasdisrupted with 700 ml of lysis buffer (150 mM trishydroxy-methylaminomethane (Tris, pH 8.0); 100 mM NaCl; 100mM ethylenediaminetetracetate (EDTA)). The fraction ofnon-detached cells was also washed twice with PBS and lysedby addition to the plate of 700 ml of lysis buffer. Both cellfractions were joined and the whole cell lysate was treatedwith proteinase K (500 mg/ml) for 2 h at 558C. Afterwards,samples were exposed to RNase A (50 mg/ml) for 16 h at378C. The DNA was first extracted with phenol, followed byphenol/chloroform/isoamylalcohol (25:24:1) and a chlo-roform/isoamylalcohol (24:1) phase. Subsequently, theDNA was precipitated overnight at y208C in 2.5 volumesof 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 waswashed with 70% ethanol. Samples were dried using aSAVANT Speed Vac Concentrator and then resuspended indistilled water. The DNA concentration was calculated bydetermining the OD260. Electrophoresis of DNA (10 mg/well) was performed for 16 h at 75 V in 1.8% agarose gelwith TAE (40 mM Tris–acetate, 1 mM EDTA, pH 8.0) asrunning buffer. The bands were visualized by ethidium bro-mide staining for 16 h and UV trans-illumination. DNA bandswere analysed by laser densitometry using a MolecularDynamics densitometer.

2.6. Quantitative evaluation of apoptosis

Briefly, Pam 212 and Pam-ras cells (105 cells/plate) wereplated at 378C in P60 sterile plates that contained cover slipsattached to the plate surface by incubation with polylysine.After cell attachment and removal of medium, the cells wererinsed three times with PBS and then treated with equitoxicconcentrations of the compounds dissolved in DMEMmedium (IC90 for 3, 10, 24, 48, 72 and 96 h). At the end ofthe incubation, the cells were fixed with 4% paraformalde-hyde in 0.1 M phosphate buffer pH 7.4 (PB) for 10 min atroom temperature, followed by acid–alcohol for 10 min aty208C, and then stained with propidium iodide (0.05 mg/ml in PBS) containing 100 mg/ml RNase A for 30 min at378C to visualize the nuclei [24,25]. The cells attached tothe cover slips were mounted on slides, counted by directvisualization through a Zeiss universal fluorescence micro-scope and photographed with Tri-X film rated 400 ASA.Apoptotic nuclei were easily distinguished from normalnuclei; they were condensed, brightly fluorescent and oftenfragmented. The results shown are means"SD of quadru-plicate independent experiments.

2.7. Protein extraction and western blot analysis

Expression of H-ras protein was detected by western blotanalysis as described previously with minor modifications

[26]. Briefly, Pam-ras cells (105 cells/plate) were treatedwith the IC90 of the compounds for 3, 10 and 24 h. Aliquotsof cell lysates containing 10 mg of total protein weresubjectedto SDS–12% polyacrilamide gel electrophoresis under dena-turing conditions. Gels were transferred to nitrocellulose fil-ters by using an electroblotting apparatus (Millipore). Thefilters were blocked with a PBS solution containing 3% (wt./vol.) of milk powder, 2% (wt./vol.) of glycine and 0.1%(vol./vol.) of Tween 20 and incubated first with anti-H-rasantibody, a monoclonal antibody against murine H-ras pro-tein (Oncogene Science), and then with a secondaryantibodyconjugated with peroxidase (goat anti-mouse IgG, Flukadiagnostics). Chemiluminiscence was performed by ECL(Amersham) according to the manufacturer instructions.Thefilters were then exposed to X-ray film and the autoradio-graphs were analysed by laser densitometry using a Molec-ular Dynamics densitometer.

3. Results

The structures of compounds 2, 3, 4 and 5 and the p-is.TSCN ligand, 1, are depicted in Fig. 1.

3.1. Analysis of the cytotoxic properties of the metallatedp-is.TSCN compounds

We have previously reported that compounds 2, 3, 4 and5 not only exhibit a cytotoxic activity similar to that of cis-DDP in several cell lines sensitive to this drug but also thatthey are active in cis-DDP resistant cell lines [18,19]. Inorder to know the specificity of the cytotoxic activity of thesecompounds against cis-DDP resistant tumour cells we havecompared the IC90 values in normal Pam 212 cells versusthose obtained in their transformed cis-DDP resistant Pam-ras cells after drug-treatment periods of 24 and 96 h.

Table 1(a) shows the IC90 values obtained after a 24 htreatment with compounds 2, 3, 4, 5, the p-is.TSCN ligand,1, and the antitumour drugs cis-DDP, etoposide, adriamycinand taxol in Pam 212 cells versus H-ras transformed Pam-ras cells together with their in vitro therapeutic index (T.I.).It can be observed in Table 1 that compounds 3, 4 and 5exhibit cytotoxic activity against Pam-ras cells at drug con-centrations significantly lower than those at which they aretoxic in Pam 212 cells because they exhibit T.I values of 1.60,1.80 and 2.10, respectively. In contrast, the cytotoxic activityagainst these cells of the clinically used drugs cis-DDP, eto-poside, adriamycin and taxol is poor because they kill Pam-ras cells at drug concentrations higher than those at whichthey induce toxicity in Pam 212 cells (T.I. values of 0.54,0.82, 0.61 and 0.75, respectively). Although the p-is.TSCNligand, 1, and compound 2 show important cytotoxic activityagainst Pam-ras cells in relation to cis-DDP (IC90 values of135 and 62 mM, respectively, versus an IC90 value of 200mM for cis-DDP) they are quite toxic in Pam 212 cells atsimilar drug concentrations since their T.I. values are 1.17



Fig. 1. Structure of compounds 1, 2, 3, 4 and 5.

Table 1IC90 mean values"SD and in vitro therapeutic indexes (T.I.) for the Pd-and Pt-thiosemicarbazone compounds against Pam 212 and Pam-ras cellsafter 24 h (a) and 96 h of treatment (b)

Compound IC90 (mM) T.I.

PAM 212 PAM-ras

(a) 24 h1 158"4 135"4 1.172 68"2 62"3 1.093 80"4 50"2 1.604 198"7 110"5 1.805 85"2 40"1 2.10cis-DDP 108"6 200"7 0.54Etoposide 152"3 184"5 0.82Adriamycin 130"5 210"8 0.61Taxol 90"2 120"4 0.75

(b) 96 h1 53"5 48"2 1.102 20"2 15"2 1.333 17"1 13"2 1.304 67"5 58"6 1.155 18"3 10"1 1.80cis-DDP 49"2 74"5 0.66Etoposide 56"4 78"3 0.72Adriamycin 32"3 82"6 0.39Taxol 27"2 35"4 0.77

Fig. 2. Cytotoxicity curves obtained in Pam-ras cells after treatment withcompounds 1, 3, 4, 5 and cis-DDP for 24 h.

and 1.09, respectively. Similar results were obtained in Pam212 and Pam-ras cells after a 96 h treatment with the com-pounds; however, the IC90 values were on the average 3–4times lower than those obtained for a 24 h incubation period(Table 1(b)).

3.2. Induction of apoptosis in Pam-ras cells by themetallated p-is.TSCN compounds

It has been reported that the ability of certain anticancerdrugs to achieve a significant therapeutic indexdifferentiatingnormal cells from malignant cells may be associated with the

fact that they induce apoptosis in tumour cells at drug con-centrations significantly lower than those needed to kill nor-mal cells [10,27]. In order to know whether compounds 3,4 and 5 (which were the ones that showed a better in vitrotherapeutic index) induce apoptosis in Pam-ras cells wechose for our apoptosis experiments the IC90 since this drugconcentration assures extensive cell killing at equitoxic dosesof the compounds (Fig. 2).

The results of the gel electrophoresis of the DNA extractedfrom control Pam-ras cells and Pam-ras cells treated with theIC90 of the compounds for 24 h are shown in Fig. 3. Just asexpected, it was observed that the DNA of control Pam-rascells appears as a band corresponding to genomic DNA (Fig.3, lane 2). Similar results were obtained in cells treated withthe IC90 of compound 1 (Fig. 3, lane 3). Interestingly, how-ever, a strong ‘DNA ladder’ indicative of apoptosis wasobtained in Pam-ras cells treated with the IC90 of compounds3 and 5 for 24 h (Fig. 3, lanes 4 and 5, respectively). Incontrast, after incubation of the Pam-ras cells with compound4 and cis-DDP, the DNA appears as a smear (Fig. 3, lanes 6and 7, respectively).

The results of the gel electrophoresis of the DNA extractedfrom control Pam 212 cells and Pam 212 cells treated with



Fig. 3. Agarose gel electrophoresis of DNA extracted from control Pam-rascells (lane 2) and Pam-ras cells treated with the IC90 of compound 1 (lane3), compound 3 (lane 4), compound 5 (lane 5), compound 4 (lane 6) andcis-DDP (lane 7). Lane 1: Hind III digested phage DNA.l

Fig. 4. Agarose gel electrophoresis of DNA extracted from control Pam 212cells (lane 2) and Pam 212 cells treated with the IC90 of cis-DDP (lane 3),compound 4 (lane 4), compound 3 (lane 5) and compound 5 (lane 6). Lane1: Hind III digested 29 DNA.f

Fig. 5. Percentage of Pam-ras apoptotic cells vs. incubation time at the IC90 of compounds 3, 4, 5 and cis-DDP.

the IC90 of cis-DDP and compounds 3, 4 and 5 for 24 h areshown in Fig. 4. It may be observed that, in contrast with thatobserved in Pam-ras cells, all the compounds produce a smearof DNA in Pam 212 cells.

Quantification of apoptosis was performed after stainingwith propidium iodide and visualization by fluorescencemicroscopy of Pam-ras and Pam 212 cells treated with thecompounds for seveal periods of time. Fig. 5 shows the per-centage of apoptotic Pam-ras cells as a function of the periodof incubation with compounds 3, 4, 5 and cis-DDP. It wasobserved that after 3 h of incubation with the IC90 of com-pounds 3 and 5, 30 and 40% of apoptotic cells are produced,respectively, with 75% of the cells becoming apoptotic after

24–28 h. The percentage of apoptotic cells decreased after 28h of treatment becoming 5% after 96 h. In contrast, treatmentof Pam-ras cells with compound 4 or cis-DDP did not induceapoptotic cells at any of the periods of time tested. Moreover,it was observed that compounds 3, 4, 5 and cis-DDP wereunable to produce the morphological changes characteristicof apoptosis in Pam 212 cells in agreement with the DNAladdering results (data not shown).

These data suggest that in Pam-ras cells the higher cyto-toxic activity of compounds 3 and 5 relative to compound 4and cis-DDP may be due to a specific induction of apoptosis.



Fig. 6. Morphological changes observed in Pam-ras cells treated with the IC90 of compounds 3 and 5 for 10 h (d and f, respectively) and photographed undera phase contrast microscope at a magnification of =200: (a) control untreated cells, (b) cells treated with 5% ethanol, (c) cells treated with the IC90 ofcis-DDP, (e) cells treated with the IC90 of compound 4.

Fig. 7. Fluorescence microscopy of control Pam-ras cells and Pam-ras cellsafter treatment for 24 h with the IC90 of cis-DDP for compounds 3 and 5.

3.3. Changes in Pam-ras cells morphology induced bycompounds 3 and 5

It has been previously reported that, in the cells growingin the monolayer, the drug-induced apoptosis leads tochanges in cell morphology and subsequent cell detachment[3,27]. In order to assess whether the apoptosis induced bycompounds 3 and 5 in Pam-ras cells produces the above-mentioned effects we performed phase contrast microscopyof control and compound-treated cell cultures. It may beobserved in Fig. 6 that, while Pam-ras cells control Pam-rascells treated with 5% ethanol, Pam-ras cells treated with theIC90 of cis-DDP and Pam-ras cells treated with the IC90 ofcompound 4 for 10 h appear as a monolayer of long cellsattached to the plate surface (Fig. 6(a), (b) and (c), (e),respectively), treatment of the Pam-ras cells with the IC90 ofcompounds 3 and 5 for 10 h induces the formation of clustersof rounded cells which are detached from the plate (Fig.6(d), (f), respectively). Moreover, we also examined thechanges in cell nuclei morphology after treatment with com-pounds 3 and 5 by staining with propidium iodide and cellvisualization using fluorescence microscopy. It may beobserved in Fig. 7 that, in contrast to cis-DDP, compounds 3and 5 induce condensed and fragmented nuclei indicative ofapoptosis in Pam-ras cells.

All these data suggest that the killing of Pam-ras cells bycompounds 3 and 5 occurs via apoptosis in agreement withthe ‘DNA laddering’ data reported above.

3.4. Levels of H-ras expression in Pam-ras cells treatedwith compounds 3 and 5

Since the Pam-ras cells used are transformed murine ker-atinocytes overexpressing the H-ras oncogene [19] we haveanalysed whether the induction of apoptosis by compounds3 and 5 is associated with a change in the levels of H-rasprotein. Fig. 8(a) shows the pattern of expression of wildtype ras protein in untreated Pam 212 cells and of H-ras



Fig. 8. (a) Decrease in the levels of expression of H-ras protein in Pam-rascells after treatment with the IC90 of compounds 3 and 5 for 24 h. Lane 3:control untreated Pam-ras cells; lane 4: compound 3; lane 5: compound 5;lane 6: cis-DDP; lane 7: p-is.TSCN ligand, 1. Lane 1: control peptide H-ras(2 mg); lane 2, control ras protein in untreated Pam 212 cells. (b) -Actina

levels from untreated and treated cells under conditions as in (a). (c)Decrease in the levels of expression of H-ras protein in Pam-ras cells aftertreatment with the IC90 of compounds 3 and 5 for 3 or 10 h. Lanes 4, 5: 3 htreatment with compounds 3 and 5, respectively; lanes 6 and 7: 10 h treatmentwith compounds 3 and 5, respectively. Lane 1: control untreated Pam-rascells; lanes 2 and 3: treatment with compound 4 for 3 and 10 h. (d) -Actina

levels from untreated and treated cells under conditions as in (c).

protein in Pam-ras cells untreated and treated with the IC90

of the compounds for 24 h. As expected, untreated Pam-rascells overexpress the H-ras protein relative to the expressionof the ras wild type protein in Pam 212 cells (lanes 3 and 2,respectively). The densitometric analysis of the bands of H-ras protein indicated that the Pam-ras cells have levels of H-ras protein about 10 times higher than that of the ras proteinfrom Pam 212 cells. Interestingly, compounds 3 and 5 pro-duce in Pam-ras cells a drastic decrease in the levels of H-ras protein relative to the control untreated Pam-ras cells(lanes 4 and 5 versus lane 3). A densitometric analysis of thegels indicated that the levels of H-ras protein in Pam-ras cellstreated with compounds 3 and 5 is 10-fold lower than thosefound in untreated Pam-ras cells and similar to those foundfor ras protein in the untreated Pam 212 cells. However,treatment of Pam-ras cells with cis-DDP or the p-is.TSCNligand, 1, did not produce changes in the levels of H-rasexpression relative to control untreated Pam-ras cells (lanes6 and 7, respectively).

Fig. 8(b) shows that the effects of compounds 3 and 5 onthe levels of H-ras protein observed in Fig. 8(a) are specific

since both compounds do not change the levels of -actina

relative to the control untreated cells and cells treated withcis-DDP or the p-is.TSCN ligand, 1 (lanes 1 to 7).

Moreover, Fig. 8(c) shows that compounds 3 and 5 alsoinduced a decrease in H-ras expression after 3 and 10 h ofincubation with Pam-ras cells, this decrease being higherafter 3 h of drug treatment (lanes 4, 5, versus lanes 6, 7).However, compound 4 did not produce a decrease in thelevels of H-ras protein after 3 or 10 h of cell treatment relativeto control untreated Pam-ras cells (lanes 2, 3 and 1,respectively).

Fig. 8(d) shows that the effects of compounds 3 and 5 onthe levels of H-ras protein observed in Fig. 8(c) are alsospecific since both compounds did not change the levels of

-actin relative to the control untreated cells and cells treateda

with compound 4 (lanes 1 to 7). Therefore, these resultsindicate that there is a relationship between the induction ofapoptosis by compounds 3 and 5 and the decrease in the levelsof H-ras protein, suggesting that most of the inhibition ofexpression of H-ras occurs within the first 3 h of drugtreatment.

4. Discussion

In the present paper, we have analysed the cytotoxicity andinduction of apoptosis by novel cyclometallated complexesof p-isopropylbenzaldehyde thiosemicarbazone and theirdimeric chloro-bridged derivatives in murine keratinocytestransformed by the H-ras oncogene which are resistant to cis-DDP (Pam-ras cells). The cytotoxic assays clearly show thatcompounds 3, 4 and 5 have specific cytotoxicity against Pam-ras cells while that of the antitumour drugs cis-DDP, etopo-side, adriamycin and taxol is non-specific. These cytotoxicitydata are noteworthy in view of the fact that etoposide, adria-mycin and taxol are drugs used in the treatment of severaltumour types currently associated with alterations in rasoncogenes [28–30]. Although the p-is.TSCN ligand showssignificant cytotoxic activity against Pam-ras cells its cyto-toxicity is non-specific since p-is.TSCN is also quite toxicagainst normal Pam 212 cells. Thus, the results obtained fromthe cytotoxicity testing indicate that compounds 3, 4 and 5have specific cytotoxic properties different from those of cis-DDP and the p-is.TSCN ligand.

We have also studied whether the higher cytotoxic activityand the better in vitro therapeutic index shown by compounds3, 4 and 5 in relation to cis-DDP are due to a specific inductionof apoptosis in Pam-ras cells. The results show that a classicaloligonucleosome ‘ladder’ indicative of apoptosis is obtainedafter a 24 h treatment of Pam-ras cells with the IC90 of theplatinated compounds 3 and 5, while a smear of DNA isobtained after treatment with the IC90 of the palladated com-pound 4 and cis-DDP. Furthermore, it is interesting to notethat treatment of Pam 212 cells with compounds 3, 4, 5 andcis-DDP induce a DNA smear. The results obtained by flu-



orescence microscopy after staining of the cells withpropidium iodide confirm the DNA laddering data and indi-cate that the percentage of apoptosis increases with the periodof incubation of Pam-ras cells with compounds 3 and 5,reaching a plateau of 75% after 28 h. However, the percentageof apoptotic Pam-ras cells decreases at higher periods of drugtreatment, becoming 5% after 96 h. In contrast, it is note-worthy that compound 4 and cis-DDP do not produce mor-phological changes associated with apoptosis in Pam-rascells. Since, in addition, compounds 3 and 5 do not seem toinduce apoptotic death in Pam 212 normal cells, it is mostlikely that the good in vitro therapeutic index shown by thesecompounds may be related to their ability to produce a stronginduction of apoptosis in malignant Pam-ras cells at low drugconcentration, as has been previously reported for other drugsin other tumour cell line types [10,27].

We have previously reported that compounds 3 and 5 formDNA interhelical crosslinks [18,19], a type of DNA adductthat is not formed by cis-DDP. So, it is tempting to thinkabout the possibility that this particular DNA lesion might beassociated with the induction of apoptosis by these novel Pt-thiosemicarbazone compounds in cis-DDP resistant cells.However, since trans-DDP (the isomer of cis-DDP) is alsoknown to form DNA interhelical crosslinks but is biologicallyinactive we think that the interhelical crosslinks formed bythe Pt-thiosemicarbazone compounds must be different fromthose formed by trans-DDP [31]. On the other hand, com-pound 4 which is the palladium analogue of compound 5 doesnot induce apoptosis in Pam-ras cells. It is likely that the lackof apoptosis induction by compound 4 may be related to thehigher lability of the DNA adducts formed by palladiumrelative to platinum [32].

Taking into account that Pam-ras cells are transformedmurine keratinocytes overexpressing the H-ras oncogene[19] we have also studied whether the induction of apoptosisby compounds 3 and 5 is associated with changes in the levelof expression of H-ras protein. The western blotting datashow that both compounds produce a drastic decrease in thelevel of the H-ras protein in Pam-ras cells being on average10-fold lower than that found in untreated Pam-ras cells andsimilar to the level of ras protein found in untreated Pam 212cells. Since a high inhibition in the levels of H-ras protein isalready present after 3 h of incubation with these platinumcompounds it is likely that the induction of apoptosis in Pam-ras cells by compounds 3 and 5 is associated with a decreasein the level of H-ras protein. Thus, due to the fact that acti-vation of H-ras oncogene induces uncontrollable cell division[33], we think that inhibition of H-ras overexpression inPam-ras cells may be a pre-requisite for sensitization of thecells to undergo apoptosis.

In summary, the results reported in this paper suggest thatplatinum compounds 3 and 5: (i) may be considered aspotential antitumour agents in view of their activity againstcis-DDP resistant cells and (ii) may probably have lowtoxic effects due to their ability to kill tumour cells byapoptosis.

5. Abbreviations

cis-DDP cisplatin, cis-diamminedichloroplatinum(II)trans-DDP transplatin, trans-diamminedichloro-

platinum(II)DMEM Dulbecco modified Eagle’s mediumEDTA ethylenediaminetetracetateFCS foetal calf serump-is.TSCN p-isopropylbenzaldehyde thiosemicarbazoneMTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-

tetrazolium bromideSD standard deviationSDS sodium dodecylsulfateTris trishydroxymethylaminomethane

Acknowledgements

This work has been supported by Grant SAF96-0041 fromCYCIT and BIO-096-0405. We thank Johnson-MattheyChem. Ltd. for its generous gift of K2PtCl4, and Dr Ramon y´Cajal, Clinica Puerta de Hierro, Madrid, Spain, for the Pam212 and Pam-ras cells. An institutional grant from Fundacion´Ramon Areces is also acknowledged. E.I.M. has a predoctoral´fellowship from the Asociacion Espanola Contra el Cancer´ ˜ ´(AECC).

References

[1] R.E. Ellis, J. Yuan, H.R. Horvitz, Annu. Rev. Cell Biol. 7 (1991)663–698.

[2] H. Steller, Science 267 (1995) 1445–1449.[3] J.A. Hickman, Cancer Metastasis Rev. 11 (1992) 121–139.[4] C. Dive, A.H. Wyllie, in: J.A. Hickman, T.R. Tritton (Eds.), Frontiers

in Pharmacology, Blackwell, Oxford, 1993, pp. 21–56.[5] G.T. Williams, Cell 65 (1991) 1–2.[6] J.F.R. Kerr, A.H. Wyllie, A.R. Currie, Br. J. Cancer 26 (1972) 239–

257.[7] M.J. Arends, A.H. Wyllie, Int. Rev. Exp. Pathol. 32 (1991) 223–254.[8] A.H. Wyllie, J.F.R. Kerr, A.R. Currie, Cell death, the significance of

apoptosisin, in: G.H. Bourne, F.J. Danielli, K.W Jeon (Eds.), Aca-demic Press, New York. Int. Rev. Cytol. 8 (1980) 251–306.

[9] P.M. O’Connor, K.W. Kohn, Semin. Cancer Biol. 3 (1992) 409–416.

[10] S.W. Lowe, H.E. Ruley, T. Jacks, D.E. Housman, Cell 74 (1993)957–967.

[11] C.M. Sorenson, M.A. Barry, A. Eastman, J. Natl. Cancer Inst. 82(1990) 749–755.

[12] M.A. Barry, C.A. Behnke, A. Eastman, Biochem. Pharmacol. 40(1990) 2353–2362.

[13] D.L. Evans, C. Dive, Cancer Res. 53 (1993) 2133–2139.[14] K.W. Kohn, R.A.G. Ewig, L.C. Erickson, L.A. Zwelling, in: E.C.

Friedberg, P.C. Hanawalt (Eds.), DNA Repair. A Laboratory Manualof Research Procedures, Part B, Marcel Dekker, New York, 1981, pp.379–401.

[15] A.M.J. Fichtinger-Schepman, J.L. van der Veer, J.H.J. den Hartog,P.H.M. Lohman, J. Reedjik, Biochemistry 24 (1985) 707–713.

[16] A.L. Pinto, S.J. Lippard, Biochim. Biophys. Acta 780 (1985) 167–180.



[17] A. Eastman, Cancer Cells 2 (1990) 275–280.[18] 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) 1399–1408.[19] A.G. Quiroga, J.M. Perez, I. Lopez-Solera, E.I. Montero, J.R.´ ´

Masaguer, C. Alonso, C. Navarro-Ranninger, J. Inorg. Biochem. 70(1998) 117–124.

[20] S.C. Dhara, Indian J. Chem. 8 (1970) 193–194.[21] R. Sanchez-Prieto, J.A. Vargas, A. Carnero, E. Marchetti, J. Romero,´

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

235–243.[22] S. Tsuchida, K. Sato, Crit. Rev. Biochem. Mol. Biol. 27 (1992) 337–

384.[23] M.C. Alley, D.A. Scudiero, A. Monks, M.L. Hursey, M.J. Czerwinski,

D.L. Fine, B.J. Abbott, J.G. Mayo, R.H. Shoemaker, M.R. Boyd,Cancer Res. 48 (1998) 589–601.

[24] M.D. Jacobson, J.F. Burne, M.P. King, T. Miyashita, J.C. Reed, M.C.Raff, Nature 361 (1993) 361–365.

[25] B.A. Barres, I.K. Hart, H.S. Coles, J.F. Burne, J.T. Voyvodic, W.D.Richardson, M.C. Raff, Cell 48 (1992) 344–355.

[26] A. Benito, M. Silva, D. Grillot, G. Nunez, J.L. Fernandez-Luna, Blood˜ ´87 (1996) 3837–3843.

[27] V.N. Sumantran, M.W. Ealovega, G. Nunez, M.F. Clarke, M.S.˜Wicha, Cancer Res. 55 (1995) 2507–2510.

[28] J. Aisner, J. Lee, Cancer 67 (1991) 215–219.[29] S.E. Lipshultz, S.D. Colan, R.D. Gelber, A.R. Perez-Atayde, S.E.´

Sallan, S.P. Sanders, New Engl. J. Med. 324 (1991) 808–812.[30] A.Y. Chang, in: W.P. McGuire, E.R. Rowinsky (Eds.), Paclitaxel in

Cancer Treatment, Marcel Dekker, New York, 1995, pp. 273–279.[31] M. Leng, V. Bravec, in: K. Hemminki, A. Dipple, D.E.G. Smuker,

F.F. Kadlubar, D. Segerback, H. Bartsch (Eds.), DNA Adducts, Iden-¨tification and Biological Significance, International Agency forResearch on Cancer, Lyon, 1994, pp. 339–348.

[32] N. Farrel, in: R. Ugo, B.R. James (Eds.), Transition Metal Complexesas Drugs and Chemotherapeutic Agents, Kluwer, Boston, 1989, pp.143–167.

[33] J.D. McGrath, D. Capon, D. Goeddel, A. Levinson, Nature 310(1984) 644–655.

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