Europ. ,7. Cancer Vol. 5, pp. 553-557. Pergamon Press 1969. Printed in Great Britain

The Role of the Carrying Molecule in the Cross-Resistance against Sugar Alcohol Derivatives

I~VA G/~TI and I. PIROSKA HORVATH Research Institute of Oncopathology ; National Cancer Institute,

Budapest, XII., Rdth Gy6rgy u.2.Hungary

INTRODUCTION EXPERIMENTAL turaours rendered resistant to one of the alkylating compounds, e.g., mus- tards, ethyleneimines or alkylsulphonic esters, have been often found to be cross-resistant to all other types of alkylating agents [1, 2]. Detection of elew~ted levels of non-protein sulphhydryl groupl~ in some resistant tumour tissues suggested that a deactivation of the alkylating agent by interaction with sulph- hydryl groups may be involved in the mecha- nism of the cross-resistance [3-5].

On the other hand, in our previous investi- gations NK lymphomas and Yoshida sarcomas rendered resistant to the sugar alcohol deriv- ative mannitol mustard (Degranol) or dibromo- dulcitol showed re.sistance to all the hexitol derivatives: mannitol mustard, dibromodulcitol, dibromomannitol and mannitolmyleran, but remained sensitive to some other types of alkylating agents, namely to TEM, trenimon and Myleran [6, 71]. It seemed to be possible that in the case of alkylating sugar alcohol derivatives resistance might be developed against both the carrying molecule itself and the alkylating function of the agent.

In order to approach this problem the poss- ible role of the length of the carbon chain was studied by means of two dibromotetritols, 1,4-dibromo-l,4-dideoxy-DL-threitol and 1,4- dibromo-l,4-dideoxy-meso-erythritol contain- ing a four-carbon chain like Myleran. The

Submitted for publication 16 May 1969. Accepted 27 June 1969.

553

role of the hydroxyl groups of the carrying molecule was studied on the 1,6-dibromo- hexane, the hydroxyl-free analogue of dibromo- hexitols (Fig. 1).

MATERIAL AND METHODS Mannitol mustard (Degranol), dibromodul-

citol and dibromomannitol (Myelobromol) and mannitolmyleran (Mannogranol) were kindly supplied by L. Instit6ris from Pharma- ceutical Factory, Chinoin; dibromothreitol and dibromo-meso-erythritol by Dr. T. Horv~tth from Pharmaceutical Research Institute. Dibromo- hexane and Myleran were commercial re- agents.

Four sublines of Yoshida ascitic tumours, resistant to mannitol mustard (Degranol) (Y-DR), to dibromodulcitol (Y-DBDR), dibro- mohexane (Y-DBHR) and dibromothreitol (Y-DBTR), were developed by a previously reported procedure [8].

The treatment started with the adminis- tration of insufficient doses and was continued with gradually increasing doses during several passages. As a result of this treatment we observed a prolongation of the survival time of the animals and a decrease of the total number of tumour cells. After the 20-25th passages, however, in spite of high range therapeutic doses the total number of tumour cells and the survival time proved to be the same as in the case of the untreated animals, showing that a resistance manifested itself to the compound.

554 l~va Gdti and L Piroska Horvdth

EOHPOUND$ TE6TED fOR C O05,, c- RE, I,,CTANEE

qn -NH- @a fu,-8, cur, c sr UO-C# #C-OH #O-C# I/O-C#

I I I I ! #0-C# nO-E# HO-E# #O-E# C~

• g n e t I I i l HC-O# #O-C# lie-on #C-O# C~

I I I I I #E-O# #E-O# nC-OH t/C-O# C~

I I I I I C#~-NH-C#,C~-Ct t~-Br C~- ~r C#,-O-SO, C#~ C~- Br

NANN/TOL HUXTA#D DI~QOHO- ~91B~OHO- ltAHNITOL- DIB#ONO- (DE6R//NOL R) DULCITOL H4NNITOL HYLERdlV IIEXANE

D OBO DBH Hie DB#

f#,-sr n o - c # no-on c#~

I I I #0- cn #c-o# c#L

I t 1 c#~- #r cu~- Br C~- o-~o,- c~

b/8#o#o-#gso- ~IS#O~tO-DL- hlYL£#AN

E~YT//#/TOZ T/I~EITOL

DBE DBT H

FIG, l .

Daily doses of the compounds were adminis- tered i.p. between the following limits:

Mannitol mustard 1-10 mg/kg Dibromo-dulcitol 10-200 mg/kg Dibromohexane 5-50 mg/kg Dibromo-DL-threitol 8-50 mg/kg

A dose of 5 × 10 ~ cells derived from the resis- tant ascites tumours was transplanted in the left dorsal region of Wistar rats weighing 150 g. Cells derived from the sensitive ascites tumour line were inoculated on the right side.

One day following transplantation daily treatment of the animals with a multiple of the therapeutic dose of the compounds was initia- ted. Drugs were administered intraperitone- ally as aqueous solutions or suspensions with Tween 80.

On the 8th day following transplantation the bilaterally growing tumours were extirpated and weighed. The tumour inhibitory effect of each compound was tested on thirty animals.

RESULTS Comparison of the growth inhibitory effect

of the compounds on bilaterally growing sensitive and resistant Yoshida tumours is presented on Table 1. The tumours of sensitive Yoshida ascites tumour line were particularly responsive to DBD, DBT and Degranol. DBM, MM and Myleran also exerted an inhibitory effect of about 50 7o. In experiments reported by Instit6ris et al. [9] DBH (6 × 15 mg/kg) did

not exert any antineoplastic effect on Yoshida tumours, and the chronic treatment with DBE (6×20 mg/kg) induced a tumour growth inhibition of only 39 7o- Increasing the dose of DBH to 5 × 50 mg/kg we could observe a slight tumour growth inhibition, whereas in the case of DBE an augmentation of the dose to 5 × 50 mg/kg did not lead to an increase of the inhibitory effect. DBH and DBE which exert a slight effect on the growth of sensitive Yoshida tumours did not show any effect on the corresponding resistant lines.

Table 2 presents the ratio of the wet weight of bilaterally growing sensitive and resistant tumours treated with the compounds tested. The symbol ' + + ' indicates the cases where the resistant tumours weighed more than twice the sensitive ones, and ' + ' where only half as much again of the sensitive ones. The symbol ' - - ' presents cases where the drugs inhibited the growth of the resistant tumours to the same degree as that of sensitive tumours.

The Yoshida tumour lines Y-DBDR and Y-DR were found to be cross-resistant to Degranol, DBD, DBM and MM but remained sensitive to DBE, DBT and to Myleran. On the other hand, the tumour line (Y-DBTR) retained full sensitivity to DBD, DBM, Degranol or MM, and proved to be cross-resistant to Myleran and DBE.

An incomplete cross-resistance, marked by ' + ' on Table 2, was found between DBH and the hexitol derivatives. The R/S ratios leading

The Role of the Carrying Molecule in the Cross-Resistance against Sugar Alcohol Derivatives

Table 1. Comparison of the growth-intdbitory effect of the compounds on bilaterally growing Yoshida tumours sensitive and resistant

Compounrl Sensitive* Y-DBDRI" DY-DR]* wet Y-DBHR$ Y-DBTR t (mg/kg) mmours weight in g

555

Without 11"94-0.23 12.6+0.81 12"34-0.35 12"5-4-0.13 12"24-0-81 treatment DBD 5×200 2.34-0.13 10.94-0.23 10.24-0.11 8.1 4-0.27 2.14-1.12 DBH 5x200 5.64-0.59 12.1+0.41 12.24-0.07 8.524-0.23 5.74-1.04 DBM 5×50 6.84-0.39 9.84-1.02 9-74-0.72 11.9 4-1-03 5.94-1.01 D 5 × 1 0 4-34-0.37 11.44-0.12 10.74-1.13 7.9 4-0.61 4.14-0.07 Mm 5×300 5.74-0-79 12"24-0.92 11.64-1.03 10.624-0.22 5.64-0.83 DBT 5x 50 3.84-0-13 3.14-0.24 3.9+0.218 4.1 -4-0.3 12.94-0.08 DBE 5×50 7.14-0.72 5.94-0.83 7.14-0.19 6.9 4-0.71 13.94-0.042 M 5× 10 5.84-0.56 6.34-0.41 6-14-0.31 6-2 4-0.01 12.24-0.03

*Average value from 80 tumours. tAverage value from 20 tumours. SAverage value from 40 turnouts.

Standard deviation ex - - -- ss X

£S S 2 s ~ = - - - - s ~ = - - X2-26

aV--1 aV

where x= individual value, ex= sum of individual values, N = number of data, s~=square of deviations, s~ = mean deviation.

to ' + ' call for some explanation. Comparing the R/S ratios with the wet weight data of Table 1, it is evident that DBH treatment was without any effect on the DBH-resistant tumour and the ratio 1,7 is a result of the slight effect of the compound on the sensitive tumour. On the other hand, in the cases of DBM, Degranol and M M treatment of the DBH resistant tumour and in the case of DBH- treatment of DBDR or Degranol-resistant tumours the ratios 1,4-1,8 are the result of a moderate inhibitory effect of these compounds on the resistant tumours.

The DBH-resistant tumour showed no cross- resistance to Myleran and to dibromotetritols.

DISCUSSION Our results point to the decisive role of the

carrying molecule in the mechanism of resis- tance to alkylating sugar alcohol derivatives. Tumours rendered resistant to a compound containing a six-carbon chain showed cross- resistance to other related six-carbon com- pounds carrying various functional groups.

The tumours with acquired resistance to dibromo-DL-threitol proved to be cross-resistant

Table 2. Cross-resistance between cytostatic sugar alcohol derivatives

Y-DBDR Y-DR Y-DBHR Y-DBTR Compounds R/S R/S R/S R/S

DBD + + + + + + (4.7) (4-3) (3.5) (0.9)

DBM + + + + + 2.1 2.1 1-5 1.0

DBH + + + 1.4 1.4 1.7 0.8

D + + + + + 2.6 2.2 1.8 0.9

Mm + + + + + 2.0 2.0 1-6 0.9

DBT -- -- + +

0"8 1 "0 1.0 3.3 DBE + +

0"8 0"9 0"9 1 "9 M + +

1.0 1.0 1.0 2.1

R/S = ratio of the wet weight of the bilaterally growing resistant and sensitive tumours.

556 ff, va Gdti and L Piroska Horvdth

to the other compounds of the four-carbon series. No cross-resistance could be demon- strated between the six-carbon ~nd four-carbon series.

Inactivation of the agent by removal of the functional groups may be generally involved in the resistance to alkylating agents. Our results suggest that the removal of the functi- onal groups of the four-carbon compounds may occur on another pathway than those in the case of six-carbon compounds.

The problem of cross-resitance to sugar alcohol derivatives becomes complicated by the fact that these compounds are present in biological systems both in cyclized and in intact forms.

Formation of epoxides from mannitolmyle- ran, dibromomannitol and dibromothreitol in vitro, was reported by Davis and Ross [10]. The 1,2-5,6-diepoxides of mannitol and dulcitol were isolated by Jarman and Ross [11]. In animals treated with mannitolmyleran or DBM Elson and co-workers demonstrated a complex blood response pattern showing both Myleran- like and epoxide-like features. These complex response patterns could be reproduced by treatment with a combination of Myleran and 1,2-5,6-dianhydro-D-mannitol. Myleran-like ef- fects seems to be derived from the intact form of mannitolmyleran and DBM [12].

In the course of our recent investigations the presence of 1,2-5,6-dianhydrohexitol and other alkylating metabolites could be detected in the serum and ascitic fluid of rats treated with DBD, DBM or mannitolmyleran [13].

It is possible that resistance has to develop both to the cyclized and to the intact forms of the drugs.

One of the possible mechanisms of resistance

to the intact form of the halogen-containing derivatives is the hydrolytic cleavage of the C-halogen bonds. In our previous experiments the C-halogen bond splitting capacity of sensitive and resistant (Y-DBDR and Y- DR) tumours was investigated. Using DBM as substrate, the extent of degradation of C-Br bond was measured by the bromide formation. Tumours resistant to Degranol and DBD showed a larger capacity to split the C-Br bond of DBM than the sensitive ones [14]. These tests were carried out in Krebs-Ringer phosphate buffer where no conversion of DBM to diepoxide occurs.

In the case of the hydroxyl-free compound dibromohexane no epoxide formation can occur. The incomplete cross-resistance of the Y-DBHR tumour line to the hexitol derivatives may be due to an inactivating mechanism which can attack the C-halogen bound but inactive against the cyclized forms of these agents.

A particular mechanism for inactivation of four-carbon compounds may be given by the cycloalkylation demonstrated by Roberts and Warwick [15]. In the case of the 1,4-dimesyl- oxy-butane (Myleran) both ends of the four- carbon chain of the compound react with the same sulphydryl group forming a cyclic com- pound. A similar mechanism may be supposed in the case of the four-carbon containing sugar alcohol derivatives DBT, DBE and their epoxide derivatives.

Admowledgements--The authors thank Dr. L. Insti- t6ris, Chinoin Factory for Pharmaceutical and Chemical Products, Budapest, for gifts of dibromodulcitol, Myelo- bromol, Mannogranol and Degranol, and Dr. T. Hor- v~th, Pharmaceutical Research Institute, Budapest, for gifts of dibromo-threitol and dibromo-erythritol.

RESUME Les auteurs gtudient les rapports entre la structure chimique et le mgcanisme de rgsistance croisge envers des ddrivls cytostatiques de polyalcools. Le role de la mol&ule porteuse a dtg Itudid sur les tumeurs de Yoshida rendues rlsistantes aux dJrivgs hexitol, Dggranol et dibromodulcitol, au dgrivd tgtritol, dibromothrditol, ainsi qu'au dibromohexane.

L'effet antitumoral des divers dgrivgs de polyalcools a gtg examing sur les tumeurs d#ivJes de la lignde sensible, ainsi que des lignges rlsistantes du sarcome de Yoshida, et inoculges bilatgralement chez l'animal.

Les tumeurs rgsistant au Dggranol ou au dibromodulcitol ont manifestg une rgsistance croisde aux autres dgrivgs hexitol. Les tumeurs rgsistant au dibromothreTtol ont montrg une rJsistance crois& envers le dibromogrythritol et le Myleran. Une rgsistance croisde incomplkte a Ltg trouv& entre le dibromohexane et les dgrivls hexitol. On ne peut dgmontrer aucune rgsistance croisge entres les composgs gt six et ~ quatre atomes de carbone.

SUMMARY The role of the carrying molecule in the mechanism of cross-resistance against cytostatic sugar alcohol derivatives was studied on Yoshida ascitic tumours which were rendered resistant to the hexitol derivatives Degranol, dibromodulcitol, to the tetritol-compound

The Role of the Carrying Molecule in the Cross-Resistance against Sugar Alcohol Derivatives 557

1,4-dibromo-l,4-dideoxy-DL-threitol as well as to the hydroxyl-free analogue of dibromo- hexitols, dibromohexane. The growth inhibitory effects of various sugar alcohol derivatives were investigated on bilaterally growing solid tumours derived from the sensitive as well as

from the resistant lines. The Degranol and DBD resistant tumours proved to be cross- resistant to other alkylating hexitol derivatives. The Yoshida tumours resistant to dibromo- threitol were cross-resistant to dibromo-erythritol and to Myleran. An imcomplete cross-resistance was found between dibromohexane and the hexitol derivatives.

No cross-resistance could be detected between the hexitol and tetritol derivatives.

ZUSAMMENFASSUNG Die Rolle des entscheidenden Molekiils in dem Resistenzmechanismus gegen alkylierende

guckeralkoholabk6mmlinge wurde gegen Degranol, Dibromodulcitol, Dibromothreitol und Dibromhexan an resistenten Yoshida Tumoren untersucht. Die antineoplastische Wirksam- keit der Substanzen wurde an bilateral gewachsenen sensitiven und resistenten Tumoren gepriift.

Die degranol- und dibromodulcitolresistenten Tumoren erwiesen sich mit anderen alkylierend, m Hexitolverbindungen als kreuzresistent. Die dibromothreitolresistenten Tumoren waren kreuzresistent mit Dibromomeso-erythritol und Myleran. Eine unvoll- standige Kreuzresistenz wurde zwischen Dibromhexan und den Hexitolabkgmmlingen gefunden. Zwischen den Hexitol- und Tetritolreihen konnte keine Kreuzresistenz nachgewiesen werden.

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