Znt. J . Cancer: 22, 558-563 (1978)

EFFECT OF RICIN A N D ABRIN ON SURVIVAL OF L1210 LEUKEMIC MICE A N D ON LEUKEMIC AND NORMAL BONEMARROW CELLS 0ystein FODSTAD and Alexander PIHL Norsk Hydro's Institute for Cancer Research, Montebello, Oslo 3, Norway

The effect of r i c h and abrin on the survival of mice treated w i t h L1210 leukemic cells intraperitoneally or intravenously was studied. I n mice given 1 x lo5 L1210 leukemia cells intraperitoneally a single dose of r i c h (2.1 pg/kg) intraperitoneally gave the best results, an increased life span (ILS) of 59%. Abrin also increased the life span of such animals although t o a lesser extent. The effect of r i c i n was superior t o that of 5-fluorouracil, b u t inferior t o that of adriamycin, which gave a maximum ILS of 280%. In mice given L1210 cells intravenously no increase i n life span was obtained w i t h ricin, abrin o r adriamycin, whereas 5-flUOrOuracil gave an ILS of 40.50%. I n spleen colony assays the differential effect of ricin and abrin on the proliferative capacity of normal hematopoietic and leukemic colony-forming cells i n bone marrow was studied. The differential effect of r i c i n was as good as that of adriamycin and considerably better than that of 5-fluorouracil. A b r i n had a much smaller effect than r i c h on both normal and leukemic cells. The effect o f abrin on the leukemic cells was too small t o be of therapeutic value. The results warrant exploration of the use o f ricin in the treatment of human leukemia.

Ricin and abrin are two highly toxic plant lectins which possess cancerostatic properties (Lin et al., 1970; Olsnes et al., 1974). Although the two toxins are isolated from the seeds of unrelated plants, they are very similar in structure and mechanism of action. The biochemical and biological properties of these toxins have been extensively studied (for review see Olsnes and 'Pihl, 1976; Olsnes and Pihl, 1978). In previous papers we have reported that abrin inhibits the growth of several transplantable murine tumors, as well as that of some human cancers growing in athymic mice (Fodstad et al., 1977). With ricin the growth-inhibiting effect was similar, although in most cases somewhat less pronounced (unpublished data). Importantly, even high doses of toxins gave no demonstrable suppressive effect on the blood level of leukocytes, indicating that the toxins have little effect on the normal bone marrow (Fodstad et al., 1977). Here we have studied the effect of ricin and abrin on the survival of mice inoculated with L1210 leukemic cells. In addition, we have measured, in spleen colony assays, the effect of the toxins on the proliferative capacity of normal and leukemic bone-marrow cells. The results demonstrate that ricin has a marked selective effect on leukemic bone-marrow cells, as good as that of adriamycin.

MATERIAL AND METHODS

Mice Female DBA/2 mice (7.to 8 weeks) weighing 19 to 22 g were used in the LCFU assay. BBD1/F1 mice of both sexes, 6 to 8 weeks old, weighing 20-25 g, were used in the other experiments. The animals were purchased from The Laboratory Breeding and Research Center, GI. Bomholt gaard, Ry, Denmark. The mice were housed eight per cage and had access to pellet food and water ad libitum. Chemotherapeutic agents Abrin and ricin were extracted from the seeds of Abrus precatorius and Ricinus communis and were purified to homogeneity as described earlier (Olsnes and Pihl, 1973a and 1973b). Adriamycin was obtained from Pharmitalia, Milan, Italy. 5-fluorouracil was a gift from Hoffmann La Roche, Basel, Switzerland. The LDIo doses after singledose intravenous administration were found to be 20 mg/kg for adriamycin, 245 mg/kg for 5fluorouracil and 0.7 and 2.8 pg/kg for abrin and ricin, respectively. After single-dose intraperitoneal administration the LDSo doses were 20 mg/kg for adriamycin, 195 mg/kg for 5-fluorouracil, 0.95 pg/ kg for abrin and 3.2 pg/kg. for ricin. The drugs were diluted to give concentrations so that the desired dose was administered in a volume of 0.2 to 0.3 ml per mouse. Tumor lines L1210 lymphoid leukemia cells had been obtained from NIH, Bethesda, Md. in 1974 and were serially transplanted twice weekly as an ascitic tumor in DBA/2 mice. The L1210 cells were implanted into test mice according to the protocols of Drug Research and Development, Division of Cancer Treatment, National Cancer Institute (Geran et al., 1972). One hundred thousand L1210 cells in 0.1 ml solution were injected intraperitoneally (L1210 ascites) or intravenously (L1210 leukemia).

Survival measurements Treatment started 24 h after implantation of the tumor cells. The drugs, in the desired concentrations, were injected using the same route as for the cells. Treatment doses and schedules are given in the Tables. Each treatment group consisted Received: August 1, 1978.

L1210

EFFECT OF RICIN ON

of 6 to 8 mice and the untreated control groups of 16 to 24 mice. All the mice were weighed on days 1 and 5 and observed for death at 1 2 - h intervals. Anti-tumor activity was expressed as the mean percentage increase in life-span (ILS) relative to that of the untreated controls. Spleen colony assays

Our assay for leukemic colony-forming units (LCFU) was carried out essentially as described by Bruce and van der Gaag (1963) and Alberts and van Daalen Wetters (1976). On day 0, lo6 L 1 2 1 0 cells were injected into the tail veins of the appropriate number of mice, and the animals

559

LEUKEMIA

15 to 20 colonies per spleen) were injected into the tail veins of groups of recipient mice. Eight days later (on day 13) the spleens were removed and fixed in Bouin’s solution. The macroscopic colonies trere counted and the average number in the drugweated animals was expressed, relative to that in the untreated control animals, as the fraction of surviving LCFU per femur. The curves shown in the charts are the results of several different experiments with one control group in each experiment. Normal bone-marrow colony-forming units (NCFU) were assayed in the same way as LCFU, except that the bone-marrow cells were injected into irradiated mice. Groups of two mice were

TABLE I EFFECT OF VARIOUS CYTOSTATIC AGENTS ON THE SURVIVAL TIME OF MICE WITH LIZ10 ASCITES ’

Drug

Ricin

Schedule

Day 1 only

Days 1 , 2 Days 1, 7

Abrin

Daily, days 1-5 Days 1 , 3, 5, 7, 9 Days 1, 5, 9 Days 1, 2, 3 Day 1 only

5-fluorouracil

Day 1 only

Adriamycin

Day 1 only

Dose

Increase in life span

Single

Total

0.85 1 .o 1.6 1.7 2.1 2.5 3 .O 1.25 1.25 1.7 2.1 0.4 0.6 0.85 0.85 0.25 0.275 0.5 0.625 45 60 100

17f0.6 28f10.3 303~2.6 35b5.7 59k4 30k9.8

-1.7 -1.4 -1.7 -2.7 -2.2 -4.7

44k7.8 25k7.6 4054.8 27k8.2 2955.1 35h6.6 32f2.2

-4.6 +0.3 -2.2 -2.8 -3.1 -2.5 -1.7

Toxic

2.5 2.5 3.4 4.2 2.0 3.0 2.55 2.55

Toxic 0.7h6.9 0.1 1-6.6 30k 11.8 22k14 39f3.4 2916.2 442r3.4 155 5 2 7 282k32

5 3

10

Weight change ( g )

( %)

-0.7 -0.1 -0.7 -1.7 1.1 0.8 -0.2 +0.9 -0.5

1 The mice were given IP injections of 1 x lo6 L1210 leukemia cells suspended in 0.1 ml of 0.9 % NaCl solution. The cytotoxic agents were also given IP. - a pg per kg body weight. - * mg per kg body weight. - SEM.- Decreased life span, i.e. shorter than that of the untreated controls.



were divided into groups of 2 mice each. One group was used as a control, while the other groups were treated on day 2 with one of the following drugs: adriamycin (3-10 mg/kg), 5-fluorouracil (25-100 mg/ kg), ricin (0.9-3.5 ,ug/kg) or abrin (0.1-0.6 pg/kg). The agents were injected intravenously in volumes of 0.2-0.3 ml. On day 5, all the mice were killed, femurs were isolated and the bone-marrow cells were washed out with medium 199 (Gibco, Glasgow, Scotland). Appropriate dilutions (sufficient to give

injected intravenously on day 0 with different drugs in the desired concentrations, and one group was left untreated. Seventy-two hours later (on day 3) the mice were killed and bone-marrow cells in appropriate dilutions were injected intravenously into groups of eight whole-body-irradiated mice (660 rad. from a Siemens “ Stabilipan” source). Eight days later (on day 11) the spleens were removed and fixed in Bouin’s solution. The macroscopic colonies were counted and the data expressed

560

FODSTAD AND PIHL

as in the case of LCFU, i.e. as the fraction of surviving NCFU per femur. RESULTS

Effect of ricin and abrin on survival of tumor-bearing mice The effect of ricin and abrin on the life span of mice given L1210 leukemic cells intraperitoneally and intravenously was measured after treatment with increasing doses given according to different schedules. For comparison 5-fluorouracil and adriamycin were included. The results are summarized in Tables I and 11. In all groups treated with ricin and abrin a signifi-

In the mice given L1210 cells intravenously (Table 11) no increase in life span was found after treatment with either r i c h or abrin. Adriamycin likewise gave no effect, while 5-fluorouracil gave an ILS of 4040%. These latter results are in agreement with those of previous investigators (Venditti, 1975). Spleen colony assays

The effect of the drugs on the normal and leukemic bone-marrow cells was measured by spleen colony assays. The results are shown in Figures 1 and 2. The dose-response curves were all linear in semilogarithmic plots. It is seen (Fig. 1, right panel) that ricin had a strong effect on the leukemic cells, while that on the normal bone-marrow stem

TABLE I1 EFFECT OF VARIOUS CYTOSTATIC AGENTS ON THE SURVIVAL TIME OF MICE INOCULATED INTRAVENOUSLY WITH L1210 LEUKEMIC CELLS ' Dose Drug

Increase in life span

Schedule Single

( %)

Total

Weight change (g)

~~

Rich

Day 1 only

Abrin

Days 1, 2, 3 Day 1 only

5-fluorouracil

Day 1 only

Adriamycin

Day 1 only

0.85 1 .o 1.6 1.7 2.1 2.5 0.85 0.25 a 0.275 0.5 60 I00

2.55

5 s 10

-2.5k3.8 9.4&9.8 1054.9 3.0k6.0 2.057.9 - 27510.1 3.1 1 9 . 8 2.5rt5.7 -0.8k7.4 - 31 +14 41 k 3 . 3 56&3.2 1.0i-2.1 8.0k6.3

-0.1 -0.6 -2.5 -2.0 -3.2 -3.5 -2.5 -0.1 -1.7 -2.4 +0.9 -0.2 +0.1 -0.1

The mice were given IV injections of 1 x 10' L1210 leukemia cells suspended in 0.1 ml of 0.9% NaCl solution. The cytotoxic agents were also given IV. pg per kg body weight. - mg per kg body weight. - SEM.

'

cant weight loss was observed. In the mice given the L1210 cells intraperitoneally ricin gave a definite increase in the life span (Table 1). The ILSvaried in the different groups from 17 to 59%. After single doses of ricin a clear dose-effect response was seen with maximum effect (ILS 59%) after a dose of 2.1 pg/kg. With a higher dose the life span again decreased, probably due to a toxic effect which is reflected in the weight loss of the animals. Abrin likewise increased the life span of the animals, although to a lesser extent than ricin. In fact, abrin did not give an ILS in excess of 30112%. With the 5-fluorouracil doses used the results were slightly inferior to those obtained with ricin. By far the best effect was obtained with adriamycin which gave an ILS of about 280% after a single dose of 10 mg/kg, an effect considerably greater than that reported by previous investigators (Goldin and Johnson, 1975; and Venditti, 1971);

cells was much smaller. With adriamycin (left panel), similar results were obtained. The magnitude of the differential effect is given by the efficacy ratio, i.e. the ratio of the slopes of the curves (Alberts and van Daalen Wetters, 1976). In the case of ricin the D,lz-value (the dose of drug which reduces the survival of the cell population by a factor of 2) was 1.8 ,ug/kg for the normal bonemarrow cells (NCFU) and 1.1 ,ug/kg for the leukemic bone-marrow cells (LCFU). The corresponding values for adriamycin were 8 mg/kg and 4.75 mg/ kg. Thus, the efficacy ratios were virtually identical, viz. 1.70 and 1.68 for ricin and adriamycin, respectively. The effect of abrin was considerably less than that of ricin, both on leukemic and on normal cells (Fig. 2, right panel). The results indicate that the LCFU would be decreased only to about 20% by an LDhodose (0.7 ,ug/kg). It is seen that large doses of abrin decreased the survival of the

EFFECT OF RICIN ON

L1210

561

LEUKEMIA

FIGURE 1 -Dose-response curves for adriamycin (left panel) and ricin (right panel) for normal (N) and leukemic (L) colony-forming units (CFU) in the mouse spleen colony assay system. Each point represents the mean & SE for eight mice.

o

10-~

NCFU LCFU 5

A

A

10

NCFU LCFU 1

Dose adriamycin (mg/ kg)

normal bone-marrow cells only to about 70% of the control value, and that even lethal doses gave no further decrease in NCFU. It is apparent from Figure 2 (left panel) that 5-fluorouracil had a strong effect on leukemic cells, but that the effect on normal cells was almost as strong and that consequently the efficacy ratio is only slightly above 1.

-

o

..

LCFU I

I

2

3

Dose rich ( p g / b )

DISCUSSION

The present results demonstrate that ricin enhanced the survival of animals given the L1210 cells intraperitoneally. An optimal ILS of 59% was found, a value well above the limit for recornrnendation as a candidate for clinical trials (Geran et al., 1972). The effect of rick was better than

LCFU 1

I

I

562

FODSTAD AND PIHL

that of abrin and also slightly better than that of 5-fluorouracil, but definitely inferior to that of adriamycin. The latter result is surprising in view of the spleen colony assays. If an estimate of the tumor-cell kill is made on the basis of the survival data, good agreement is obtained with the LCFU data in the case of ricin, abrin and 5-fluorouracil. However, in the case of adriamycin the cell kill estimated from the survival data was much greater than that observed in the spleen colony assays. We have no adequate explanation for this apparent inconsistency. However, it should be noted that the ILS here observed after treatment with adriamycin is considerably greater that that found by previous investigators (Goldin and Johnson, 1975; Venditti, 1971). In fact, the cell kill calculated from the ILS observed in the latter studies is in good agreement with that found in the present spleen colony assays. R i c h and abrin failed to enhance the survival of animals given the L1210 cells intravenously. This is hardly surprising as these proteins would not be expected to pass the blood/brain barrier. Adriamycin, which is known to be unable to pass the blood/brain barrier, likewise failed to enhance the survival of animals given the L1210 cells intravenously. On the other hand, 5-fluorouracil which does pass into the central nervous system, increased significantly the life span of such animals. Although 5-fluorouracil increased the survival of animals given L1210 cells intravenously, the spleen colony assays showed that the drug had a low differential effect on leukemic cells. Therefore, the initial beneficial effect on survival observed in this rapidly growing tumor will presumably be offset by delayed effects on the normal bone-marrow cells. The survival data alone may therefore be misleading. The present results suggest that, before a drug found active against L1210 ascites is considered for clinical use against leukemia, it should be tested also in spleen colony systems. In the present tumor model ricin was found to be more efficacious than abrin, judged both from the survival data and from the spleen colony assays. In contrast, in other murine tumors and certain human tumors growing in athymic mice we have found abrin to be more efficacious (Fodstad et al., 1977). Thus, in spite of the similarities between abrin and rich with regard to structure and biochemical mechanism of action (Olsnes and Pihl, 1976, 1978) the effect of the two toxins on different

cell types may differ. For reasons which are not apparent, leukemic and normal bone-marrow cells appear to be considerably more sensitive to rich than to abrin. Abrin had too small an effect on leukemic cells to be of therapeutic value. Thus, an LD,, dose decreased the LCFU only by a factor of five. However, the spleen colony assays demonstrated that doses of abrin which are effective against certain other tumors suppress only slightly the growth potential of normal bone-marrow cells. This corroborates our previous finding in other tumor models that growth-inhibiting doses of abrin had no depressive effect on the peripheral leukocyte counts. Since most other active anti-cancer drugs in current use have a strong bone-marrow depressive effect, the slight effect of abrin on the normal bone marrow could render this drug useful in combination therapy (Fodstad et al., 1977). Both for the normal hematopoietic and the leukemic colony-forming cells, exponential survival curves were obtained in the spleen colony assays with no saturation at the doses employed. Such data have been interpreted to mean that the agents kill cells in all or most portions of the generation cycle (Bruce et al., 1966). In this case the interpretation is supported by the finding that synchronized HeLa cells were found to be equally sensitive to abrin and rich in all phases of the cell cycle (Olsnes and Refsnes, 1978). The reason why the leukemic cells seem to be inherently more sensitive to ricin than the bone-marrow cells is not clear. Possibly the mechanism involves increased binding and uptake of the toxin in the leukemic cells. The most interesting finding in this work is that ricin has an appreciable differential effect on leukemic bone-marrow cells, an effect similar in magnitude to that of adriamycin, a drug which is currently used in the therapy of human leukemia. The data suggest the possibility that ricin, like adriamycin, may be of value in the treatment of human leukemias and lymphomas in combination with other drugs, including those which are effective against central nervous system lesions. ACKNOWLEDGEMENTS

This work was supported by The Norwegian Cancer Society. The skilful technical assistance of Mr. Roy Krokaa is greatly appreciated.

EFFET DE LA RICINE ET D E L’ABRINE SUR LA SURVIE DE SOURIS ATTEINTES DE LEUCEMIE ~ 1 2 1 0ET SUR DES CELLULES DE MOELLE OSSEUSE LEUCEMIQUES ET NORMALES Les auteurs ont 6tudi6 l’effet de la ricine et de l’abrine sur la survie de souris trait& avec des cellules leuc6miques L1210 en intraptritonkale ou en intraveineuse. Chez les souris qui avaient reGu 1 x lo5cellules L1210 en intraperitoneale, c’est une dose unique de ricine (2.1 pg/kg) en intrapbitonbale qui a donnt les meilleurs rtsultats, une duree de vie accrue (ILS) de 59 %. L’abrine a aussi prolong6 la vie de ces animaux, mais dans une moindre mesure. L’effet de la ricine etait superieur B celui du 5-fluorouracile, mais infkrieur B celui de l’adriamycine, qui donnait I’ILS maximum, 280 %. Chez les souris qui avaient r e p des cellules L1210 en intraveineuse, on n’a pas observe d’augmentation de la dur6e de vie sous l’effet de la ricine, de I’abrine ou de l’adriamycine, mais, avec le 5-fluorouracile, I’ILS 6tait de 40-50%. Dans les essais de colonies splhiques, on a btudib l’effet differentiel de la ricine et de l’abrine sur la capacitC proliferative des cellules hematopoietiques normales ou des cellules leuctmiques provenant de la moelle osseuse qui forment des colonies. L’effet de la ricine 6tait aussi bon que celui de l’adriamycine et bien meilleur que celui du 5-fluorouracile. L’abrine avait un effet beaucoup moins fort que la ricine, tant sur les cellules normales que sur les cellules leucemiques. L’effet de l’abrine sur les cellules leucemiques etait trop faible pour avoir une valeur therapeutique. Les resultats justifient des recherches sur I’emploi de la ricine dans le traitement de la leuctmie humaine.

EFFECT OF RICIN ON L 1 2 1 0 LEUKEMIA

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REFERENCES

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OLSNES,S., and PIHL,A., Different biological properties of the two constituent peptide chains of ricin, a toxic protein inhibiting protein synthesis. Biochemistry, 12, 31 21-3126 (1 973a). OLSNES,S., and PIHL,A., Isolation and properties of abrin: a toxic protein inhibiting protein synthesis. Europ. J . Biochem., 35, 179-185 (19736). OLSNES,S., an a PIHL,A., Abrin, r i c h and their associated agglutinins. In: P. Cuatrecasas (ed.), The specificity and action of animal, bacterial and plant toxins, pp. 131-173, Chapman and Hall, London (1976). OLSNES, S., and PIHL,A., Abrin and ricin-two toxic lectins. Trends biochem. Sri. 3, 7-10 (1978). OLSNES,S., and REPSNES,K., O n the mechanism of toxin resistance in cell variants resistant t o abrin ana ricin. Europ. J. Biochem., 88. 7-15 (1978). OLSNES,S., REFSNES,K., and PIHL, A., Mechanism of action of the toxic lectins abrin and ricin. Nature (Lond.), 249, 627-631 (1974). VENDITTI,J. M., Treatment schedule dependency of experimentally active antileukemic (L 1210) drugs. Cancer Chemother. Rep., 2, 35-59 (1971). VENDITTI, J. M., Relevance of transplantable animal-tumor systems t o the selection of new agents for clinical trial. In : Pharmacological basis of cancer chemotherapy, pp. 245270, The Williams and Wilkins Company, Baltimore (1975).

Effect of ricin and abrin on survival of L1210 leukemic mice and on leukemic and normal bone-marrow cells.

Znt. J . Cancer: 22, 558-563 (1978) EFFECT OF RICIN A N D ABRIN ON SURVIVAL OF L1210 LEUKEMIC MICE A N D ON LEUKEMIC AND NORMAL BONEMARROW CELLS 0yst...
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