Ind ian Journal of Ex perimental Biology Vo l. 37, May 1999, pp. 429-433

Role of bio-metal Fe(III) in anticancer behaviour of tamoxifen

Jyotsna Shukla & Krishna S Pitre

Electrochemi cal Laboratory, Department o f Chemi stry, Dr Harisingh Gour Uni versity, Sagar 470 OQ3, Indi a

Recei\'ed 15 April 1998. revised 20 Jalluary 1999

Phys icoc hemical. mi crobial and pharmacological studies on Fe (III) - T amoxifen complex have been carri ed out in solid and aqueous phases. On the basis of elemental analys is, polarographic studies, amperometric titratio ns and IR spectral ~tudies the probable fo rmula fo r the complex has been worked out to be I: I , Fe( II1) - Tamoxifen. A tentati ve structure has heen suggested to the compl ex. The metal li gand interaction has been studied usi ng polarographi c method at 27° ± 1°C and at ionic strength of 11 = 1.0 (KCI). Microbial studies on the complex was carried out against various pathogenic bacteria and rungi using Raper's method. Mouse sarcoma cell line 180 and Balb/C mi ce were used fo r the anticancer screening of solid co mpex . i ll \'i lm and ill vi vo, respecti vely. The results of microbi al and pharmacological studies with the M:Drug complex revealed that the complex is more potent as compared to the pure drug .as regard s to its anticancer activity. As such Fe (III ) Tamox iren co mpl ex may be recommended to the therapeutic experts for its possible use as more potent anticancer drug.

The biochemi cal, pharmacological and medicinal importance of metal-drug complexes is very well establi shed " :; . In continuati on to the work done in our laboratory on the study of electrochemical, bio­inorganic, mi crobi al and pharmacological behavi our of some metal-drug complexes6

.9

, the present paper deals with the said studies on the Fe(lli)-Tamoxifen (2-[p-( I ,2-diphenylbut-l-enly) phenoxy] ethyl­dimethylamine (anticancer drug) complex.

Chemical and reagents-All the chemicals used were of Analar/BDH grade. The drug Tamoxifen (C26 H29NO) was procured from Mis Lyka labs Ltd .. Bombay. Double di stilled water and absolute methanol were used as solvents.

Polarographic measurements were made on an Elico (Hydrabad) pul se pol arograph model CL-90, coup1ed with a X-Y polarocard model LR-IOS. A dropping mercury electrode (DME) as a working e lec trode, a coiled platinum wire as an auxiliary e lec trode and saturated cal omel electrode (SCE) as a reference e lec trode, were used .

Ex perimental sets were prepared by keeping over all iron (metal ion) and potass ium chloride (supporting elec trolyte) concentration fixed at 1.0 mM and 1.0 M, respectively. The ligand concentration was varied from 0 .5 to 5 mM. The pH of the test so lution was adjusted to 7.0 ± 0 .2 using NaOH solution.

The amperometric titrations were performed on a manuall y operated set-up, equipped with a polyflex galvanometer (sensiti vity S. I x I 0-9 ampJdiv.) and an

AJCO vamier potentiometer. DME was used as an indicator electrode and a calomal electrode served as reference electrode. The capillary characteristics of the DME had a m2IJ, t "6 values of 2.13 mg2l3 Sec·t12 at 50 cm effecti ve hight of mercury column .

The pH of all the test solutions was measured on an Elico digital pH meter model LI-I OS .

Experimetal sets, each having different but known amount of the drug understudy were prepared in appropriate quantity of supporting electrolyte (potass ium chloride) at pH 7 .0±0.2 were prepared and titrated separate ly against the standared solution of the titled Fe (ill) ions whose pH has been adjusted to that of the titrate (7.0±0.2).

Synthesis procedure of the solid complex-Ferric chloride and Tamoxifen (drug) solutions were separately prepared in methyl alcohol (50:50 v/v) and were mixed in I: I molar ratio. The mixture was then refluxed in a round bottom flask for one-two hours. The res idue (complex) was filtered and washed thoroughly to remove any unreacted materials. The complex was dried at low temprature (40°C) and stored over P40 JO .

The elemental , C, H, Nand 0 analysis of the complex was done on a coleman elemental anlyser model-29/33, at CDRL Lucknow, whereas gravimetric method was used for the estimation of . . h I to Iron m t e comp ex .

The IR spectrum of the solid complex was recorded using KBr pe llets on a perkin-Elmer IR spectrophotometer, model-379.

430 INDIAN J EXP SIOL, MAY 1999

BiolQgical study of Fe(/II) - tamoxifen complf~x Microbial study-Raper's paper disc method" was

followed for the microbial screening of Fe (III) -Tamoxifen complex against various bacteria, viz. Staphylococus aureus , Salmonella typhi , Bacillus pumilus and Vibreo colame and fungi : A. Jumigatus and Chrysosporium sp.

Pharmacological studies - In vitro and in vivo studies of anti-cancer activity of the prepared drug metal complex have been done by following

12·1 5 procedure . II! vitro - Mouse Sarcoma Cell Line-ISO was

obtained from National Center for cell Science, Pune, India, as a monolayer culture in Roux bottles (Corning Plastics U~A).

Cell culture - The Cells obtained were cultured in 5 ml 24 well culture plate (corning plastic USA). The

ce ll s were seeded in 2 x 105 ce ll s per well and 1.0 ml of Dulbecco's modifi ed Eagle's medium (DMEM) containing 10% (v/v) Foetal Calf serum, penicillin

100 pg/ml and streptomyc in 100 )..Ig/ml was added to each' we ll. The cell s we re kept in incubator at 37°C for 4hr in 5'7(' CO2 atmosphere and 95 % humidity . The ce ll count was made on Neubaurs chamber (Fine Optik , German y).

Three di lutions, viz. I, 10 and 100 )..IM/ml of pure drug and its iron complex was made and then the ce ll s were trea ted as follows : Column Free drug Column Metal complex

A I pM ( Iml) D I )..1M ( 1m!)

B 10 )..lM ( lml ) E 10pM ( l ml)

C 100 pM ( I ml ) F 100 )..1M (I ml) Arter additi on of the respective so lutions, the

culture plate was incubated at 37°C for 4 hr. Finally the ce ll counts we re made as under. These counts were compared with the ce ll cu lture in DMEM without treatment.

Cell viabilitv count - Ce ll viability counts were made by Trypan blue dye ex luc ion test. Two drops of trypan blue was added to each cell culture well and kept for 15 min . Now, a drop of culture was added to hemocytometer (Neu baurs Chamber) and the number of stained, non stained and total number of cell s were counted. Then, the inhibition was calculated using the equation:

No of viab le ce ll s - No. of viable ce ll s afte r treatment

-------------------------xIOO

No. of viabl e cells without treatment

The experiment with each concentration of the drug and the complex was repeated thrice and statistical conclusion were drawn .

In vivo - The comparative efficiency of pure and complex fOnTIS of tamoxifen drug was evaluated from the difference in response after treatment with the two forms of drug, using:

Animal model :Balb/c mice. Wei ght 30-40 g.

Tumor model :Sarcoma - 180

Drug :Tamoxifen and its iron complex

Cells growing in nutrient medium (DMEM) were obtained from NCCS, Pune. They were brought into single cell suspension by trypsinization (0.2% Trypsin). The ce ll suspens ion was centrifuged to

obtain concentrated suspension ( 1-2x 105 ce ll s/ml). Approximately 105 ce ll s of tumor were injected on the dosal surface of the mou se and a llowed to grow. Palpable size was reached by 6-8 days.

T he time requ ired to double the tumor volume (volume doubling time [VDT] ) from 100 to 200 mm' was taken as a criterion to assess the antitumor effic iency of pure and complex dru g in S-180 tumor bearing mice. The trea tment was started after tumor size reaches 100± 10 mny' . Indicated dose (equivalent to 0 .2 mg) of free drug and drug complex were inj ected interavenously and tu mor growth was monitored . Tumor s ize was calcu lated by the formul a 1/2 LW 2

. Where L-Iong diameter and W- short diameter of the tumor. The above in vivo excperiment was repeated on two other sets of mice groups.

Acute toxicity (LDso)-The acu te toxi ci ty of the complex was determined in mice (6 mice, 3 male and 3 female). Animals were fasted for 12 hr prior to the test and then compl ex was admini stered . I 16 IIlte ravenous y .

Results and Discussion Polarographic hehavior of tamox:i/ell with Fe

(I/I}-In 1.0 M KCI at pH 7.0 ± 0.2, Fe (III) and its complex with the li gand understudy were found to be reversibly reduced involving three electrons. Which was evidenced by the plotes of log i/(id-i) Vs E. The reduction was found to be diffu sion contro ll ed, which

was evidenced by the plot of iJ Vs . './hcnrr .

On gradual increase of the Tamoxifen concentration the half wave potential of Fe (III) me tal ion shifted to more e lec tronegti ve value and the

SHUKLA & PITRE: ROLE OF BIO-METAL Fe(III) IN ANTICANCER BEHAVIOUR OF TAMOXIFEN 431

diffusion current also decreased, thereby showing complex formation between Fe (ill) with Tamoxifen.

To study the composition and formation constant of the complex, plots of bE II2 (shift in the E I12) i.e. bE I /2 = (E lI2h against log C. (logarithm of the concentration of the ligand) were drawn. The plots were linear lines showing the formation of single complex species in solution. Lingane treatment l 7 of the observed polarographic data reveals I: I metal : tamoxifen complex formation with formation constant log ~ = 4.5 .

Amperometric determination of tamoxifen with Fe (/II) -Fe (III) gives a well defined polarographic wave in 1.0 M KCI at pH 7.0 ± 0.2. The diffusion current wa.s found proportional to the concentration of Fe (III). The tamoxifen drug does not produce any wave under the said experimental conditions . The plateau potential for the polarographic wave of Fe (III) (- IAv) Vs Hg pool was applied for carrying out amperometric titration . On performing the amperometric titration of drug solution with standard solution of Fe( III), the current volume plots resulted in shaped curve. The end point as located by graphical method revea led metal to drug ratio of I: I, which is in agreement with the author's observations on the metal : li gand equilibria using polarographic method.

Characterization of Fe(/II) - tamoxifen complex Elemelltal allalvsis - The results of elemental

analysis (Table I) of the drug and it's complex with Fe(III) revealed I: I metal : drug ratio in thi s complex , which supports author's findin gs using polarographic and amperometric methods.

IR.Spectra - Structurally important IR band of the

Table I - An alytical data of tamoxifen and its complex wilh Fe (III )

Analysis/Ca lcu lated/( Found) Element Tamoxifen Fe(III )-Tamoxifen

complex

13.00

( I J.OO) 13.00

c X·UN 73 .06

(X4 0 I ) (73.13)

H 7.X I 6.55

(77J) (649 )

.1 .77 3.28

URO) (327)

o 4.3 1 3.75

(4.27) (3.69)

tamoxifen drug and its Fe(lII) complex which are particularly useful in assigning the position of metal ligand bonding is observed as a broad band at 1601 -1640 cm· 1 due to C-N aliphatic vibrations ' 8 in the IR spectrum of the drug. Which is found to be affected in the spectrum of Fe(III) - tamoxifen complex i.e. the broad band at 1600-1640 cm- I results in a single peaked band at 1590 cm· 1 with reduced intensity .19 These observations clearly indicate the involvement of the nitrogen of CoN group in complex formation ?O On the basis of above data a tentative structure of the Fe(III) - tamoxifen complex may be given as under

Microbial studies Results of antimicrobial activities of the Fe(llI) -

Tamoxifen complex are shown in Table 2. A peru sal of the data in table clearl y shows that Fe(III)­Tamox ifen complex is found to be more tox ic as compared to the control drug against al l the te: t bacteria and fun gi.

Pharmacological studies 111 \'ilro _ . The re sult s of ill \'ilro ex periment s of

pure drug and its complex are shown in Table ~. A perusal of the results show t hat Fe( 111)-Tamox i fen complex was found to be more effective than pure drug. The complex under study showed an increased inhibition against the S-1 80 tumor ce lls at all the test concentrations i.e . I. 10 and 100 ~lM/ ml. The increased inhibition activity of the complex was 60 .S ± 1.1 9r , 74.0 ± 1.89c and 91 . ~ ± 2.5 9c as against -+6.7 ± l.OCk , 67A ± 2.2 9c and 8 1.0 ± l. 5 9c shown by the drug, respectively . The :-.tatistical treatment of the obse rved inhibiti on data i.e. standard deviati on and coefficient of variance which never exceeded 1.1 and 2A 9'c respecti ve ly, speak the reliability of the observed inhibition data .

111 vivo - The results of the average of mice tumor size against tamoxifen drug and its iron complex under study suggested that the tumor volume was 0.048 cm1

, on the tumor cell inj ected mice without

432 INDI AN J EX P BIOL, MAY 1999

Table 2-Antimicrobial study of tamoxifen Fe(lII) compl~x

Test organi sm Inhibition-zone* (mm)

Conc. of complex per 10 mI

O.S mM

I. Baderial

a. Staphylococus au reus

b. Salmonell a typhi 6.4

c. Bacillu s pumilus

d. Yibreo w larae

2. F ungal

a. A fumigatu s

b. Chrysosporium sp

7.0

8.3

10.S

*Including diameter of filt er paper di sc.

1.0 mM(B)

IS.O

9.0

13.8

19.3

21.7

Tah le 3- /11 \';Im cytotoxici ty of tamoxifen and Fe( " I ) - tamoxi fen complex against S- 180 tumor cell s

Co mpound

Tam(lxifcn

Fe(lll )- Tamoxifcn complex

Concentration

~M/ml

1.0

10.0

100.0

- 1.0

10.0

100.0

(a) Co mposi te results of three ex periments. (h ) Mean + standard error of mean.

% inhibition after 4h

46.7 ± 1.0(·) (h)

67.4 ± 2.2

8 1.0 ± 1.5 60.S ± 1.1

74.0 ± 1.8

9 1.3 ± 2. S

ad mini sterin g drug . or comp lex after 20 days. The vo lu me was reduced to 0.032 cm}, on the tumor

injected mice who were a lso administered the

tamox ifen drug. However, in case of Fe(III) -

Tamoxifen complex admini stered mice (tumor cell

injected) showed s ignificant decrease in the tumor

volume of 0.02 1 c m}. Thus indicating the in vivo

tu mo r in hibiti on power of the complex over the drug

understudy over the ex perimental time peri ods i.e. 20

days.

S imil ar results were observed with the other two mice groups . However. the stati stical treatment of the observed inhibition data i.e . standard deviation and coefficient of variance for the three mice groups, which never exceeded 1.0 and 2.3%, respective ly, c learl y reveals the re li ability of the observed data. However, to judge the signi ficance of the observed data, each in-vi vo ex periment was repeated five times with the drug and the Fe(III)-tamoxifen complex; respec ti ve ly. On applying the students 't' test21 to the observed ce ll viabi I ity counts data, it revealed p vatue

Control Fe(lII) metal

(A)

1.0 mMllOml

37. 1

27.3

29.8

31.S

39.4

41.9

% change over control metal

(A-BIA)xl oo

4S.1

69 .8

S6. 1

SI.O

48.2

Contort tamoxifen drug

(Y)

1.0 mMllO mI

26.1

24.S

30.2

2!S.7

2S.4

31.6

% change over control drug

( Y-B/Y) x loo

38.7

70.2

SI.9

24.0

31.3

of < 0.0) in a ll the cases. Thus clearly indicating that the data is s ignificant.

The LD50 of the complex aga inst mice was found to be 210 mg/kg body weight intravenoul y. The reported LD50 for tamoxifen is 200 mg/kg agai nst mice. This shows that the complex is not tox ic and could be used fo r medicina l purpose.

From the above in vitro and in vivo results it could be concluded that tamoxi fe n complex with Fe(III) is found to be more effective to control the multiplication of sarcoma cell s as compared to the tamox ifen drug. The increased cytotoxicity of the Fe(III)-temoxifen complex may be explain on the orgument that the complex under goes in vivo hydrolys is. The hydrolysed products are react ive and capable of forming OH-bridged dime rs and trimers, resulting in enhenced destructi on of DNA con dons by the complex under study was compared to the parent drug. T he reactive compuds of Fe(III) may also bind to guanine (G) and cytocine (C) rich regions of DNA. Bes ides iron induced unbinding of DNA may also take place due to strong affinity of Fe(UJ) for guanine. 22 Thus Fe(III)-tamoxifen complex may be recommended to the therapeutic experts as a more potent anticancer drug in lieu of the drug taken for present study .

Acknowledgement The authors are thankful to Prof. S.P. Banerjee,

Head, Chemistry Department , and Prof. N.K. Jai n, Pharmacy Department , Dr. Haris ingh Gaur University , Sagar, for providing necessary laboratory faciliti es in the department respecti vely. Thanks are also due to M.P. Council of Science & Technology,

SHUKLA & PITRE: ROLE OF BIO-METAL Fe(II1) IN ANTICANCER BEHA YIOUR OF TAMOXlFEN 433

Bhopal , for the award of research fellowship to one of the authors (1S).

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