Jnt. J. Cancer: 3, 260-264(1968)

STUDIES ON CROSS-RESISTANCE TO DEGRANOL- AND DTBROMODULCITOL-RESISTANT YOSHIDA TUMOURS

EVA GAT1 Research Institute of Oncopathology, Budapest, Hungary

Cross-resistance studies were carried out on a series of sugar alcohol derivatives substituted in positions I and 6 with various functional groups. Yoshida ascites turnours resistant to Dibromodulcitol as well as to Degranol proved to be cross-resistant to Degranol, Dibromodukitol, Dibromomannitol and Mannitolmyleran. Myleran and Vinblastine sulphate inhibited the growth of the sensitive and resistant turnours to the same degree. Experiments with bilaterally growing solid forms of these tumours yielded the same result. The relationship between the chemical structure and the mechanism of this cross resistance is discussed.

The relationship between chemical structure and cytostatic effect was investigated on a series of sugar alcohol derivatives substituted in positions 1 and 6 with various functional groups. These compounds, Mannitol mustard, Degra- no1 R, Dibromodulcitol, Dibromomannitol, Man- nitolmyleran (Fig. 1) differed greatly from each other in their inhibitory effects on individual turnours (Kellner et al., 1955; lnstitoris et al.,

1963; Nemeth, 1966; Kellner et al., 1967a; Kellner et at., 19676; Palyi, 1967). Nevertheless, cross-resistance studies on these agents on N K ascitic lymphoma yielded positive results (Gati, 1966). The possibility of such cross-resistance occurring in solid turnours has now been in- vestigated. Yoshida tumour, which grows equally well in the ascitic and solid forms, was chosen as the test object.

C H 2-N H ,-C H ,-C H 2-C I I I I I

CH,-Br CH,-Br C N 2-0-C H 3-S 0

HO-C-H H-C-OH HO-C-H I I I

I I I H-C-OH HO-C-H H-C-OH

I I I H-C -OH H-C-OH H-C-OH

1 I I C H 2-N H 2-C H ,-CI CH,-Br CH2-Br

HO-C-H HO-C-H HO-C-H

HO-C-H I

I H-C-OH

I H-C-OH

I CH2-0-

HO-C-H

-CH3-S02

Degranol R Dibromodulcitol Dibromomannitol Mannitolmyleran (Mannitol mustard)

FIGURE 1 Chemical formulas for the sugar alcohol derivatives tested.

Received: 13 June, 1967. Approved: 5 July, 1967.

260

CROSS-RESISTANCE TO DEGRANOL AND DIBROMODULCITOL

MATERIAL AND METHODS

Rats bearing Yoshida ascites tumours were rendered resistant to Degranol as well as to Dibromodulcitol (DBD) by a previously re- ported procedure (Gati, 1963).

Wistar rats weighing 150g were inoculated with 5 lo6 cells of the originally sensitive Yoshida ascites tumour cells and the same dose of the forms resistant to Degranol and Dibromodulci- tol. Degranol (10 mg/kg), Dibromodulcitol (200 mg/kg), Dibromomannitol (200 mg/kg), Mannitolmyleran (300 mg/kg), Myleran (10 mg/ kg) and Vinblastine sulphate (0.3 mg/kg) each day, were administered for 5 successive days, beginning one day after the inoculation of the tumour cells. On the 8th day after transplantation the animals were sacrificed and the total volume (TV) of tumour cells was determined. The total volume of the treated group was expressed as a percentage of the control:

TV,O/;; =

ascitocrit 'i: A ascitic fluid of treated group ascitocrit O/;; x ascitic fluid of control group

(TV, treated group expressed as a percentage).

x 100

= total volume of tumour mass from the

On the 4th day following inoculation a dose

of the compounds approximating LD,, was administered to the animals bearing Yoshida ascites turnours (Degranol 100 mg/kg, Dibro- modulcitol 1,500 mg/kg, Dibromomannitol 1,500 mg/kg, Mannitolmyleran 3,000 mg/kg, Myleran 100 mg/kg, Vinblastine sulphate 3 mg/kg). Before treatment, and 6, 12, 24, 48, 72, 96 and 120 hours after treatment, the number of de- generating tumour cells, that of abnormal mitoses and the number of multinucleated turnour cells were determined by counting 500 cells in each smear.

A dose of 51( lo6 cells derived from the resistant ascites tumours was transplanted in the left dorsal region of 20 rats in each group. Cells derived from the sensitive tumours were inoculated on the right side.

One day following the transplantation, daily treatment of the animals with the therapeutic doses of the drugs was initiated. On the 8th day following transplantation the bilaterally growing tumours were extirpated and weighed.

RESULTS

Experimetits on ascites turno~rs

Figures 2 and 3 In animals treated with Vinblastine sulphate,

the total volume of both sensitive and resistant

FIGURE 2 Changes in total volume of Degranol-resistant and

-sensitive Yoshida ascitic tumour cells upon the effect of the compounds tested. Average value from 20 animals in each group.

MdNNllOl- VINBIAJJII MYltRAN JUiPUAl i DiGRaNo'

FIGURE 3 Changes in total volume of Dibromdulcitol-

resistant and -sensitive Yoshida ascitic tuniour cells upon the effect of the compounds tested. Average value from 20 animals in each group.

26 1

tumours in ascitic forms decreased by 96-99 %. Myleran was found to be equally effective o n both sensitive and resistant tumours, reducing the total volume of cells by 70% in both groups. Therapeutic doses of Degranol, Dibromodulcitol, Dibromornannitol and Mannitolmyleran re- duced the total volume of sensitive tumours by 96, 98, 87 and 67% respectively, whereas in the Dibromodulcitol- and Degranol-resistant tumours the decrease was somewhat more than 30%.

Table I

In the first 6-12 hours after a single large dose of the chemotherapeutic agents mitotic disturbances and an increase in the number of degenerating tumour cells were seen. Then came a period (24-72 hours) during which cellular disintegration was predominant and the increase in the number of degenerating cells and abnormal mitoses reached its maximum. During the next period (72-120 hours) a number of multinucleated giant cells appeared. Myleran and Vinblastine sulphate brought about equal cytomorphological changes in the sensitive and Dibromodulcitol-

resistant tumour cells. On the other hand, the sugar alcohol derivatives tested failed t o bring about essential changes in the Dibromodulcitol- resistant tumour cells. Abnormal mitotic figures occurred only to a small degree. Even the number of multinucleated cells did not rise after the treatment.

Experiments with solid Yoshida tumours

Table II The tumours of sensitive Yoshida tumours of

ascitic origin were particularly responsive to Dibromodulcitol, Degranol and Vinblastine sul- phate. The other agents-Mannitolmyleran, Dibromomannitol and Myleran-also exerted an inhibitory effect of more than 50%.

Myleran or Vinblastine-sulphate inhibited the growth o f the Degranol and Dibromodulcitol- resistant tumours to the same degree as that of the sensitive ones.

The weight of both Degranol- and Dibromo- dulcitol-resistant tumours remained unchanged after the treatment with Degranol, Dibromo- dulcitol, Dibrornornannitol and Mannitolmyleran.

TABLE I

CYTOMORPHOLOGICAL EFFECT OF THE COMPOUNDS TESTED ON YOSHIDA ASCITIC TUMOUR CELLS SENSITIVE A N D RESISTANT TO DIBROMODULCITOL

Time elapsed after treatment (hours) Before

treatment 12 24 48 72 I20 Compound

tumours AM'DC' MC3 A M D C MC AM DC MC AM D C MC A M D C MC A M D C MC

Dibromo- S' 0 12 8 2 15 7 2 31 5 4 27 9 7 39 12 3 52 42 dulcitol R5 0 10 12 0 14 4 0 8 6 0.3 7 6 0 10 4 0 I1 5

Dibromo- S 0 12 8 3 47 10 4 17 9 1 19 16 1.5 24 18 0.8 23 10 mannitol R 0 10 12 1.5 12 3 I 8 6 0 10 4 0 11 5 0.2 9 7

Degranol S 0 12 8 1 28 I I I 28 15 4 33 5 1 35 28 1 36 39 R O 1 0 1 2 I 1 4 4 0 5 5 0 8 4 0 . 5 7 3 0 9 6

Mannitol- S 0 12 8 0.7 31 7 1 29 12 2 43 14 4 33 18 5 27 23 myleran R 0 10 12 0 11 6 0.4 16 3 0 22 4 0.2 12 4 0 12 8

Myleran S 0 12 8 1 27 6 2 39 19 3.8 30 24 2 32 15 1.7 23 7 R 0 10 12 1.7 28 14 2 40 22 4 39 25 1.8 41 14 2 26 8

Vinblastine S 0 12 8 4 32 18 6 41 22 8 47 40 9 49 32 2 31 29 sulphate R 0 10 12 3 33 7 5 42 12 9 52 33 1 1 54 30 3 29 35

' Abnormal mitoses. il Degenerating cells.

Percentage o f multinucleated ct:lls based o n counting 500 cells in each smear. Sensitive tumour. Resist ant t umo u r.

262

CROSS-RESISTANCE TO DEGRANOL A N D DIBROMODULCITOL

TABLE I 1

COMPARLSON OF THE GROWTH-INHIBITORY EFFECT OF T H E COMPOUNDS O N BILATERALLY GROWING YOSHIDA T U M O U R S SENSITIVE A N D

RESISTANT TO DIBROMODULCITOL AS WELL AS TO DEGRANOL

Dibromo- Sensitive dulcitol- Degranol- tumours ‘ resistant resistant

(wet (wet (wet weight in g) weight in g) weight in g)

Before treatment . 12.75 10.2 12.4 Dibromodulcitol . 2 9.6 10.4 Dibromomannitol . 5.9 12.3 12.6 Degranol , . . . 4.4 11.4 10.5 Mannitol-myleran . 6.3 12.1 11.6 Myleran . . . . 6.8 6.1 7. I Vinblastine sulphate 4.3 3.9 4.6

~~ ~

’ Average value from 20 animals in each group.

DISCUSSION

Our results indicate a considerable degree of cross-resistance to different types of alkylating sugar alcohol derivatives in Yoshida tumours with acquired resistance to Degranol as well as to Dibromodulcitol. The cross-resistance to

other alkylating agents in a number of resistant strains of animal neoplasms is remarkable. Cross-resistance to drugs is not always due to a common metabolic alteration (Hutchison, 1965). Two possible mechanisms are often considered ; alteration of membrane permeability towards the agent and deactivation of the agent by bonding to nonprotein sulphydryl groups (Wheeler, 1963).

Our recent observations suggest a special deactivating mechanism based on dehalogena- tion in acidic environment (Gati and Horvath, 1967). In view of the low reactivity of nucleo- philic groups a t acidic p H the dehalogenating capacity of resistant tumours might be due to enzymatic hydrolysis. This type of detoxification might be involved in cross-resistance to cytostatic agents containing halogen in their functional groups.

On the other hand, Mannitolmyleran, an alkyl sulphonic acid derivative of a sugar alcohol, containing no halogen in its functional groups- in contrast with Myleran-showed cross-resist- ance in Degranol- or Dibromodulcitol-resistant tumours. This points to a possible role of the sugar molecule in the induction of this cross- resistance.

ETUDES SUR LA RESISTANCE CROISEE DE TUMEURS DE YOSHIDA RESISTANT AU DEGRANOL ET AU DIBROMODULCITOL

Des etudes de resistance croiske ont CtP faites sur une &vie de derivks d’akools de sucres, substitues en positions I et 6 a divers groupements fonctionnels. Les tumeurs ascitiques de Yoshida resistant au Dibromodulcitol et au Degranol ont manifestk une rksistance croiske au Degranol, au Dibrornodulcitol, au Dibromomannitol et au Mannitolmyleran. Le Myleran et le sulfate de vinblastine inhibaient la croissance des tumeurs sensibles et des tumeurs resistantes dans la mkme proportion. Des experiences portant sur des formes solides, a croissance bilatkrale, de ces tumeurs, ont abouti au nikme resultat. L’auteur examine les rapports entre la structure chimique et le mkcanisme de cette resistance croisee.

REFERENCES

GATI, E., Morphologische und enzymchemische GATI, E., and HORVATH, I . P., Dehalogenation of Veriinderungen an einem Mause-Asciteslymphom cytostatic sugar alcohol derivatives in tumour mit induzierter Resistenz. Z. Krebsforsch., 65, tissues. Vfh I n f . Chemother. Congr., Vienna (1967); 506-5 I2 ( I 963). Part 1, p. 459. Verlag d. Wiener Med. Akad.,

Wien resistenten NK/Ly-Ascites-Tumoren. Z. Krebs- HUTCHISON, D. J., Studies on cross-resistance and f ~ r s c h . , 68, 184-189 (1966). collateral sensitivity. Cancer Res., 25, 1581 (1965).

GATI, E., Kreuzresistenzuntersuchungen an degranol-

263

GATI

INSTITORIS, L., HORVATH, 1. P., and CSANYI, E., Activite cytostatique de certains derives halogenes de polyalcools. 111' Symp. int. Chimiothirapie, Naples, 1961; Vol. 111, p. 250-262. S. Karger, Basel (1963).

KELLNER, B., NEMETH, L., and SELLEI, C., Die biologische, hiimatologische und geschwulsthem- mende Wirkung eines neuen Stickstoff-Lost- Derivatives 1,6 - bis - (p-chlor-athylamino) - I ,6 - de- soxy-D-mannit-dichlorhydrat. Naturwissenschaft.,

KELLNER, B., NEMETH, L., INSTITORIS, L., and HORVATH, I . P., 1,6-Dibronio-I ,6-dideoxy-dulcitol: A new antitumoral agent. Nalure (London.),

42, 582-584 (1955).

213, 402-404 ( I 967~) . KELLNER, B., NEMETH, L., SUGAR, J. , CAT[, E.,

PALYi, I . , and DOBROSSY, L., Pharmacology of 1,6-Dibromo-l,6-dideoxy-dulcitol and its cyto- static effect on transplantable tumours. Arzneimit- telforsch., 17, 1037-1043 (19676).

NfiMETH, L., Vergleichende Untersuchungen von anticarcinomatosen Mitteln in Tierversuch. Fourth Hungarian Conference for Therapy and Pharmacolo- gical Research, Budapest, 1966; p. 168. Ed. Hung. Pharmacol. SOC., Budapest (1966).

PALYI, I., Effects of antitumour agents Degranol, Mannitol-Myleran, Dibrommannitol, Dibromodul- citol on cell morphology in tissue cultures. Neo- plasma, 14, 159-166 (1967).

WHEELER, P. G., Studies related to mechanisms of resistance to biological alkylating agents. Cancer Res., 23, 1334 (1963).

264