A dual anti-tumor effect of a combination of interferon-α or interleukin-2 and 5-fluorouracil on...

CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY

Vol. 62, No. 1, January, pp. 103-111,1992

A Dual Anti-Tumor Effect of a Combination of Interferon-a Interleukin-2 and 5-Fluorouracil on Natural Killer (NK)

Cell-Mediated Cytotoxicity’

ZVI REITER, OSMAN N. OZES, LAWRENCE M. BLATT,* AND MILTON W. TAYLOR

or

Z~tit~te for ~o~u~r and Cellular Biology, De~rtment of Biology, ~~d~~a University, Remington, Indian 47405; and *Amgsn Co., ~ho~and Oaks, Ca~ifo~~ 91320

Previous in vitro and in vivo studies have shown a syner- gism between interferon (IFN) and B-fluorouracil (5.FU) against different tumor cell lines. In the present study we report that the combination of IFN-cz and 5-FU has a significant effect not only on the inhibition of tumor cell growth but also on the regulation of natural killer cell- mediated cytotoxicity (NK-CMC). The addition of 5-FU to effector cell population neither a&&s NK cell activity nor activation of NK cells by IFN or by interleukin (IL)-2 How- ever, p~t~atment of target cells with 5-FU increased their su~eptibility to NK activity and abolished the protective effect induced by IFN against NK-CMC. This dual effect of IFN-a and 5-FU was found to be applicable to target cells of different origins including a cervical carcinoma cell line (ME-1801, a hairy cell leukemia-like cell line (Eskol), a CML cell line (K-5621 and a primary culture of AIDS-related Kaposi’s sarcoma cells. Similar results were found with IL-2 treatment of Eskol cells but not other cells. Combination of IL-2 with 5-FU resulted in enhancement of the sensitivity of the cells to NK activity and abolished the protection against NK-CMC. Based on these results we propose that the eom- bination of 1FN-a and 5-FU not only has a direct growth inhibit04 effect on tumor cells but also has a regulatory role on the immunological arm of the NK-CMC. Moreover, since the combination gave the same pattern of response in different tumor cells, both NK-sensitive and NK-resistant, this combination treatment may be a candidate for clinical trials in various types of tumors. 0 1992 Academic Press, INC.

INTRODUCTION

Interferon (IFN) was originally described as an antiviral agent. However, in the last few years, it has be- come evident that IFNs (esp~ially IFN-cc) have a significant anti-proliferative, i~unom~ulato~ and dif- ferentiating effect on a variety of tumors (l-4). Recombinant IFN-a is widely available for routine clinical use. However, although promising results with

lTbis research wa8 supported by U.S.P.H.S. Grant CA53783 and the American Cancer Society Grant CD-468.

IFN have been documented both in experimental ani- mals and in human tumors, such as hairy cell leukemia and Kaposi’s sarcoma, the therapeutic efficacy of IFN is still limited. One possible approach to improv- ing the potency of IFN therapy is to combine it with chemotherapeutic agents. Indeed, several studies have reported in vitro synergistic or additive interactions of IFN with the anti-neoplastic agent, 5-flurouracil (5- FU) G-101, an important agent for the treatment of adenocarcinoma of the colon, renal cell carcinoma, colorectal cancer, and other neoplastic diseases. Prelim- inary data suggest that synergy between IFN-(r and 5FU may be clinically relevant (11). In patients with colon cancer, a combination of 5-FU and IFN-o gave a response rate higher than that anticipated with 5-FU alone (12-14). In uitro studies demonstrate that the combination of IFN-cu and 5-FU has a direct antiproliferative and cytotoxic activity on tumor cells. Be- cause IFN is an immunoregulatory molecule, it is possible that this combination also affects the immune host defence mechanism including the natural killer (NK) cell system.

NK cells function as an early defense mechanism against tumor cells and their metastases. IFN plays a central role in regulation of NK activity. On the one hand, IFN increases the lytic activity of NK effector cells against sensitive target cells and induces cytotoxic activity against normally resistant target cells. On the other hand, pretreatment of target cells with IFN decreases their sensitivity to NK cell-mediated cytotoxicity (NK-CMC) (15, 16). Interleukin (IL)-2 is also a regulator of the immune response. In vitro culture of killer cells in the presence of IL-2 results in the generation of a very potent cytotoxic cell population, the l~phokine-activated killer (LAK) cells (17).

In the present study we report the effect of in vitro combination treatment of IFN-ol or IL-2 and 5-FU on NK-CMC directed against various tumor cells. It was found that incubation of NK effector cells with 5-FU had no effect on IFN-primed NK activity nor did it affect LAK generation by IL-2. However, treatment of

103

009%1229ls2 $1.60 Copyright 6 1392 by Academic Press, Inc.

All righte of reproduction in any form reserved.

104 REITER ET AL.

tumor cells with 5FU alone increased their susceptibility to NK-CMC and the combination with IFN-a abolished the protective effect induced by IFN against NK-CMC. This dual effect of IFN-cx and 5FU was found on various target cells of different origins including a cervical carcinoma cell line (ME-1801, a hairy cell leukemia-like cell line (Eskol), a CML cell line (K-5621, as well as a primary culture of AIDS-related Kaposi’s sarcoma. Like IFN, IL-2 induces activation of killer cells. IL-2 can also protect tumor cells from NK-CMC although this effect is limited only to fresh leukemic hairy cells (18) and Eskol cells. 5FU was also found to have the ability to abolish the protection against NK- CMC induced in Eskol cells by IL-2.

MATERIALS AND METHODS

Target cells. The following cells were used as targets: A cervical carcinoma cell line (ME-180, ATCC HTB33), a chronic myelogenous leukemia (CML) cell line (K-562, ATCC CCL 2431, a hairy cell leukemia- like cell line (Eskol, 191, and primary culture of AIDS- related Kaposi’s sarcoma (KS, 20). K-562 and Eskol cell were grown and assayed in RPMI-1640 medium (GIBCO Inc., Grand Island, NY) supplemented with 10% inactivated fetal calf serum, L-glutamine (2 m&f), penicillin (100 U/ml), and streptomycin (100 pg/ml). ME-180 cells were grown in McCoy’s medium and KS cells were grown in DMEM medium with the same supplements. Cells were grown at 37°C in a humidified 5% CO, incubator.

NK effector cells. Purification of effector cells was as described (16). Briefly, nonadherent human leukocytes were purified from freshly collected peripheral blood of healthy donors. The cells were fractionated by sedi- mentation on Ficoll-Paque (Parmacia LKB Biotech- nology Inc., NJ) followed by adsorption on a nylon-wool column. The nonadherent cells were used as the effector cells in the experimental procedures.

Cytokines and 5-FU. Recombinant consensus IFN-CY (sp act 2 x 10’ units/mg) and human recombinant interleukin-2 (“Ala-125,” sp act >5 x lo6 units/ mg) were provided from Amgen, Inc. (Thousand Oaks, CA). 5-FU was purchased from Sigma.

Treatment of cells with cytokines and 5-FU. Effec- tor cells (5 x lO‘?ml) were cultured in medium alone, in medium containing IFN (300 antiviral units 10.1 ng]/ml) for 18 hr at 37”C, or in medium containing IL-2 (100 units/ml) for 3 days, in order to generate lymphokine-activated killer (LAK) activity. Adherent target cells (KS and ME-1801 were seeded in flat-bottom 96- well plates (1 x lo4 cells/well) 24 hr before adding cytokines. The cells were then cultured with various concentrations of either IFN, IL-2, 5-FU, or a combination of either IFN + 5-FU or IL-2 + 5-FU for 18 hr prior to use as target cells in the various NK assays, or

for various time periods for use in the anti-proliferative assays. Eskol and K-562 cells (1 x lO?ml) were grown in suspension and treated with the above cytokines, drug, or combinations under similar conditions.

Measurement of cell proliferation. KS and ME-180 cells (1 x lo4 cells/well) were treated for varying periods of time in media containing various concentrations of IFN-(r, IL-2, 5-FU, or combinations. Following incubation, the cells were stained with crystal-violet (8% in methanol) and the dye extracted with sodium citrate (0.1 M) in 50% ethanol (100 @well). Absorbance was read at 590 nm. K-562 and Eskol cells (1 )< lo5 cellsi ml) were treated as described. Cell samples were harvested at 24-hr intervals and counted following trypan blue staining for cell viability. The growth inhibition assays expressed as the percentage increase in the number of cells in the treated cultures relative to the increase in the number of cells in nontreated cultures according to the formula: % growth = [(I, - C&C,, -.- C,)] x 100, where I, is the number of treated cells at Day n; C, is the number of control cells at Day n and C, is the number of control cells at Day 0.

5’Cr-release cytotoxicity assay for NK-CMC. KS and ME-180 cells were seeded in flat-bottom 96-well plates for 24 hr (lo4 cells/well). The cells were labeled with 51Cr-sodium chromate (2 l&i/well; sp act 393.18 mCi/mg; NEN) for 12 hr at 37°C. Following labeling, the cells were washed three times with phosphate- buffered saline (PBS). Various amounts of effector cells were then sedimented (final volume was 200 kl/wellj for 4 hr of incubation at 37°C. Aliquots of the superna-, tants were collected and radioactivity was measured in a y-counter (Beckman). The sensitivity of Eskol and K-562 cells to NK-CMC was measured similarly: cells were labeled with 51Cr-sodium chromate (300 &Wml ! for 1.5 hr. The cells were washed twice with PBS, sus- pended in media to a density of 1 x lo5 cells/ml and dispensed into 96-well conical-bottom microtiter plates (lo4 cells/well; 100 plj. Various amounts of effector cells were then added, (final volume was 200 &well) for 4 hr at 37°C. Total cpm was obtained by adding Triton X-100 (2%, 100 pi/well, Sigma) to wells with target cells alone. Spontaneous release was determined in wells containing target cells alone. Data are expressed as percentage specific release according to the formula: Specific release (%I = (cpm, - cpm,/cpm, cpm,) x 100, where cpm, are experimental cpm; cpm, are spontaneous cpm, and cpm, are total cpm. Each data point represents an average of triplicate samples.

Conjugate formation assay. Target cells were tested for their capacity to form conjugates with effector cells. KS cells were seeded in flat-bottom 96-well plates (lo4 cells/well; 100 ~1). After 24 hr of incubation effector cells were sedimented (1 x 106/well; 100 plj. The cocultures were incubated at 37°C for 60 min and

COMBINATION THERAPY OF INTERFERON AND 5-FLUOROURACIL 105

then, the cells were stained with trypan blue. The ability of Eskol and K-562 cells to form conjugates with effector cells was similarly determined: effector cells (1 x lO?ml; 100 ~1) and target cells (1 x 104/ml; 100 ~1) were mixed, centrifuged (12Og; 5 min) and incubated at 37°C for 30 min. After gentle resuspension, the cells were stained with trypan blue. At least 400 cells were counted and the number of target cells bound to two or more lymphocytes was divided by the total number of these cells, giving a percentage of conjugate formation. In addition, the number of trypan blue-stained target cells in conjugates was divided by the total number of conjugates, giving a percentage of dead cells in conjugates.

Cold target competition assay. Various amounts of unlabeled K-562 or Eskol cells were used as competitive targets for NK activity. Competitor cells were mixed with effector cells (lo6 cells/well) and incubated for 15 min at 37°C. 51Cr-labeled K-562 cells (2 x 104/ well) were then added (final volume 150 pJ/well), the plates were incubated, and released radioactivity was harvested and counted as described before.

Production of crude NK cytotoxic factor (NKCF). Cell-free supernatants containing NKCF were gen-

erated by incubating NK effector cells (2 x lo6 cells/ ml) with target cells (effector:target ratio 5O:l) in serum-free medium. Cultures of either effector or target cells alone were used as controls. After 48 hr of incubation (37°C in 5% CO,) the supernatants were collected, filtered through a 22-pm Millipore filter, and stored at - 70°C until further use.

Assay for NKCF. 51Cr-labeled target cells (lo4 cells/well, in 50 p,l serum-free media) were sedimented in 96-well plates. Supernatants containing various amounts of crude NKCF preparations were added and the cultures were incubated for 20-24 hr. Aliquots of the supernatants were then collected and total, spontanous and specific cpm were calculated as described above.

Statistical analysis. All the results are expressed as an arithmetic mean + a standard error of the mean @EM). Comparisons were carried out by a Student t test.

RESULTS

An Additive Effect of IFN and 5-FU on Cell Growth Inhibition

When a series of tumor cells, including Eskol (Fig. la), ME-180 (Fig. lb), K-562 (Fig. lc), and AIDS- related KS (Fig. Id) cells were treated in combination with IFN-ol and 5-FU, an additive inhibitory effect on cell growth was found. This effect was both time (Fig. 1) and dose dependent (not shown). The additive growth

inhibitory effect was found to be statistically significant (P < 0.01 in Eskol and KS cells and P < 0.05 in K-562 and ME-180 cells). IL-2 did not exhibit any antiproliferative effect on these cells (not shown).

5-FU Affects neither NK Cell Activity nor NK Cell Activation by IFN or LAK Generation by IL-2

5-FU has no effect on the cytotoxic ability of NK effector cells (Fig. 2a). Combination treatment of IFN-a and 5-FU resulted in levels of NK activation similar to those of IFN treatment alone (Fig. 2b). 5-FU also did not exhibit any effect on LAK generation by IL-2 (Fig. 2~). Various doses of 5-FU (0.01 ng-10 pg) were tested in these experiments. Figure 2 presents the activity of the various effector cells on K-562 target cells. The cytotoxic activity of IFN-primed NK cells was higher than that of unprimed NK cells while LAK cells exhibited the highest level of cell-mediated cytotoxicity. Similar results were obtained using other tumor cells as targets (not shown).

5-FU Increases the Sensitivity of Tumor Cells to NK-CMC

The susceptibility of NK-resistant Eskol (Fig. 3a), ME-180 (Fig. 3b), KS (Fig. 3d) target cells, and NK- sensitive K-562 (Fig. 3c) target cells to NK cell- mediated cytotoxicity was increased significantly (P < 0.01-P < 0.05) following preincubation of the tumor cells with 5-FU. The effect was found to be dose- dependent. Similarly, the sensitivity of these tumor cells to either IFN-primed NK activity or LAK activity (not shown) was increased when the target cells were pretreated with 5-FU.

5-FU Abolished the Protective Effect against NK-CMC Induced by IFN or by IL-2

Treatment of cells with IFN reduced their susceptibility to IFN-primed NK-CMC. This phenomenon was found in ME-180, KS, K-562 (not shown), and Eskol cells (Fig. 4a). The same pattern of response was found using LAK cells as effecters (not shown). Combination treatment of IFN-u and 5-FU abolished the protective effect against NK activity induced by IFN (Fig. 4a). A dose-dependent relationship in 5-FU action was found: low doses of 5-FU only increased the sensitivity of Es- kol cells to NK activity while high doses of 5-FU resulted in a significant (P < 0.05 and P < 0.01) loss of the protective effect (Fig. 4a). Similar results were obtained using the other cells as targets (not shown). A unique phenomenon was found in Eskol cells following treatment with IL-2. Both IFN- and IL-2-treated cells became more resistant to NK-CMC. Combined treatment of IL-2 and 5-FU abolished this protection against NK-CMC (Fig. 4b) in a dose-dependent manner.

106 REITER ET AL.

a - b

20 40 60 60 20 40

Time (hr)

60 60 100

FIG. 1. Growth inhibition effect of 5-FU + IFN-Con1 combination. Tumor cells at concentration of 1 x lo5 (Eskol, a; ME-160, b; K-562, c; KS, d) were cultured for varying periods of time in the presence of IFN-Conl(300 units/ml, 0); 5-FU (1 pgiml, 0) or a combination of the two drugs (IFN 300 units/ml + 5-FU 1 pg/ml, A). The growth inhibition is expressed as percentage increase in the number of treated cells relative to the increase in the number of control cells. The curves represent the data of one of two or three experiments.

5-FU Does Not Affect Target Cell-Effector Cell pretreatment of competitor cells (Eskol or K-5621 Conjugate Formation but Increases the Sensitivity changed their ability to compete for 51Cr-labeled K-562 of Tumor Cells to NKCF cells. IFN reduces the ability of tumor cells to induce

Pretreatment of Eskol cells with either IFN or IL-2 activation of effector cells and NKCF release following

reduces their sensitivity to NK cell-mediated cytotox- effector-target cell conjugate formation. 5-FU did not

icity. Combined treatment of the tumor target cells show a significant effect in this step of the killing pro-

with both IFN and 5-FU or IL-2 and 5-FU abolished cess (not shown). However, 5-FU treatment did have

this protection. We therefore investigated the mecha- an effect on the sensitivity of target cells to NKCF

nism of 5-FU action at the cellular level. Eskol cells activity. It was found that 5-FU increased significantly

were pretreated with IFN, IL-2,5-FU, or combinations (P < 0.01-P < 0.05) and in a dose-dependent manner

(IFN + 5-FU and IL-2 + 5-FU) and their capacity to the sensitivity of Eskol cells to NKCF activity (Fig. 6).

form conjugates with effector cells measured. Table 1 In this stage of killing, IFN and IL-2 are not involved.

indicates that none of the treatments changed the level Combination treatment of 5-FU with either IFN or

of conjugate formation. However, when conjugated IL-2 had the same effect as 5-FU treatment alone, i.e.,

dead cells were counted it was found that following enhancement of the susceptibility of the tumor cells to

either IFN or IL-2 treatment, the number of dead con- NKCF.

jugated Eskol cells was decreased while following 5-FU treatment more conjugated dead cells were counted

DISCUSSION

(Table 1). The cold target competition assay supported IFN has an anti-proliferative effect on several tumor this finding. As shown in Fig. 5, neither IFN nor 5-FU cells (l-41 and plays a key role in the regulation of

COMBINATION THERAPY OF INTERFERON AND 5FLUOROURACIL 107

! a

b

C

50 25 12 6

Effector cell : target cell ratio 5-FU has no effect on NK or LAK activities. Effector cells

(NK cells, a; IFN-primed NK cells, b; and LAK cells, c) were precul- tured in medium alone (m), or in medium containing 5-FU (10 rig/ml, m, 100 rig/ml, 0; 1 pg/ml, q ). K-562 cells were used as targets. The histograms represent the % of specific 51Cr release from target cells. Spontaneous 51Cr release was 5-9% and SEM was 1.3-2.8. Figure 2 represents the data of one of three experiments using effector cells from three different donors.

various immunological functions. For example, IFN can affect antibody formation, antigen presentation, cell-mediated immunity, and macrophage activation (21). IFN has also a central role in the regulation of NK activity. It increases the lytic activity of NK effector

cells against sensitive target cells and it also induces cytotoxic activity against normally resistant target cells. On the other hand, pretreatment of target cells with IFN decreases their sensitivity to NK-CMC (15, 16).

In the last few years, clinical trials of combination of IFN-a with chemotherapeutic drugs in cancer patients have been described. It was suggested that chemoim- munotherapy of tumors may involve first a reduction in the tumor load by chemotherapeutic agents. Surviv- ing tumor cells may then be removed by immune defence cells, such as the NK population, the activity of which can be increased by immunostimulators, such as IFN-a and IL-2 (22). Indeed, In vitro synergistic or additive interactions of IFN with 5FU were reported (5- 9) in preliminary clinical trials on colon carcinoma and renal cell carcinoma patients. It was found that synergy between IFN-(u. and 5-FU may also be clinically significant (11-14). Since IFN is an immunoregulatory molecule, it is possible that the combined treatment induces regulation of the immune host defence mechanisms such as the NK system. In our studies, we have found that 5FU did not affect the activity of NK effector cells, and was not involved in the priming of NK cells by IFN or the generation of LAK cells by IL-2. Charamella et al. (23) tested the effect of various clinically used chemotherapeutic agents on NK activity in vitro. They reported that 5-FU had no effect on either NK or IFN-primed NK activities at the concentrations used in clinical treatments. However, it is well documented that 5-FU has some immunosuppressive effects at both cellular and humoral levels of the immune response due to interference with purine metabolism (24). The lack of suppression of NK activity (which was tested at doses corresponding to the therapeutic levels of the drug) suggests that the NK parameter of the immune system is functional in the presence of 5-FU. It is possible that the effect of the chemotherapeutic agents on the immune system is based on drug selec- tivity. In this respect, the large granular lymphocytes associated with NK activity may possess membrane characteristics which do not allow these agents to pen- etrate into the cytoplasm and exert their immunosuppressive effect (23).

Contrary the lack of effect of 5-FU on NK effector cell level, we found that 5-FU did affect the tumor cells. Pretreatment of tumor target cells with 5-FU increased their susceptibility to NK cell-mediated cytotoxicity. This phenomenon was found in various types of tumor cells from various origins such as CML (K- 562), cervical carcinoma (ME-1801, leukemic hairy cell-like (Eskol), and AIDS-related KS cells. Our results are in agreement with the work of Ujiie (221, who has reported that treatment of EL-4 cells with 5-FU significantly increased the sensitivity of these cells to both murine NK and CTL cytolysis. In addition to its ability to induce NK activity, IFN-cx also has the

108 REITER ET AL.

60 -

d

0 10 20 30 40 50 0 IO 20 30 40 60 60

Effector cell : target cell ratio

FIG. 3. 5-FU increases NK susceptibility. Tumor cells (Eskol, a; ME-180, b; K-562, c; KS, d) were cultured for 18 hr in the presence or absence (0) of 5-FU (1 rig/ml, A; 100 rig/ml, n ; 1 *g/ml, A). Various amounts of NK effector cells were sedimented for 4 hr of incubation. The curves represent the % of specific 51Cr release from target cells. Spontaneous ‘lCr release was 5-17% and SEM was 1.3-4.8. Figure 3

represents the data of one of three experiments using effector cells from three different donors.

capacity to reduce the susceptibility of tumor cells to NK activity. We have found that 5FU can increase the sensitivity of IFN-treated tumor cells to NK-CMC. Moreover, at high doses of 5FU, the protective effect against natural killing induced by IFN, was completely abolished and the target cells became more sensitive to NK activity than control cells. This pattern of response to IFN plus 5-FU was found in all of the tested tumor cells. One can suggest that the enhanced sensitivity of tumor cells to NK-CMC and the abolishment of the IFN-protection effect may be the result of the target cells becoming “sick” following incubation with 5FU. Such a toxic effect may be responsible for an increase in the fragility of the target cell to NK cell- induced injury. However, five lines of evidence do not support the above possibility: (i) Although after 18 hr of incubation with 5-FU, a fraction of target cells do die, a significant cytotoxic effect of 5-FU is not observed until after a long period of incubation (3-4 days). (ii) The cells which were killed by 5-FU after a short period of exposure cannot incorporate radioactive chro- mium and as a result are not measured in the NK

cytotoxic assay. (iii) Although the spontanous release of 51Cr from target cells increased after treatment with 5-FU from 5-9% to 5-17% this value is taken into ac- count in calculating the specific release of 51Cr. More- over, by using relatively low doses of 5-FU (1 and 100 rig/ml), the spontanous release of 51Cr does not change significantly while the sensitivity to NK-CMC does. (iv) The protective effect of IFN against NK-CMC re- quires mRNA and new protein synthesis. It has been reported that various metabolic inhibitors such as Actinomycin D, cycloheximide, and emetine can abolish this protection (25). Moreover, it seems that tumor cells actively resist NK attack by the constitutive synthesis of certain proteins. Since 5-FU is also a metabolic inhibitor, we expect it to behave in a similar fash- ion. (v) The ability of 5-FU-treated tumor cells to bind and form conjugates with effector cells was found to be the same as in untreated cells. The cold target competition assay also supports this observation. 5-FU- treated cells did not lose their ability to activate NK effector cells following binding. Thus, we suggest that 5-FU treatment does not result in a sick cell with en-


SO

a b 40 -

30 40 50 0 10 20 30 40 50 60

Effector cell : target cell ratio FIG. 4. 5-FU abolished IFN- and IL-%-induced protection against NK activity. Eskol cells were cultured in the presence of medium alone

(O), IFN (300 unite/ml, Cl, in a), IL-2 (100 units/ml, Cl, in b) or with combination of IFN or IL-2 and 5-FU (1 rig/ml, A; 100 rig/ml, A; 1 p,g/ml, n ; 10 &ml, 0) for 18 hr. IFN-porno NK effector cells were added for 4 hr of incubation. The curves represent the % of specific 51Cr release from Eskol cells. Snontaneous 51Cr release was 7-16% and SEM was 1.8-5.4. Figure 4 represents the data of one of three experiments using effector cells from three different donors.

hanced sensitivity to NK-CMC but has a more specific effect.

Eskol cells, a leukemic hairy cell-like line exhibited a unique phenomenon. In these cells not only IFN, but also IL-Z reduced the sensitivity of the treated cells to NK-CMC. Combination treatment of 5-FU and IL-2 gave a result similar to that of 5-FU and IFN. At low doses of 5FU, Eskol cells became more sensitive to NK activity and at high doses of 5-FU the protection against NK-CMC induced by IL-Z was completely abolished. The other tested cells did not respond to IL-2 treatment.

TABLE 1 Conjugate Formation between Eskol and Effector Cells

Conjugate Conjugated Target cells formation dead cells

Effector cells treatment (W) (%)

NK 4.5 2.4 NK IFN-a= 3.9 1.6 NK IL-26 5.2 0.7 NK 5-FUc 4.1 6.3 NK 5-FU + IFN 5.0 4.7 NK 5-Fu + IL-2 4.7 5.5

IFN-an-primed NK - 5.1 22.3 IFN-a-primed NK IFN-ol 4.9 11.5 IFN-u-primed NK IL-2 4.2 9.3 IFN-u-primed NK 5-FU 5.6 34.5 IFN-a-primed NK S-FU + IFN 4.7 24.0 IFN-a-primed NK 5-FU + IL-2 6.1 21.4

-- Note. The conjugate formation assay was done in duplicate using effector

cells from three d&rent donors. SEM was * 0.63.4. s Target cells or effector cells were treated with IFN-Conl(300 units/ml) for

18 hr. *Target cells were treated with IL-2 (300 U/ml) for 18 hr. c 1 pg/ml.

A model that divides the interaction between NK cells and NK target cells into several phases has been proposed (25, 26). The NK cell first has to bind to the target cell through a receptor that recognizes a specific structure on the target cell surface. The second phase involves the activation of intracellular processes in the

10 1 , I 1 0 2 4 6 6 10 12 14 16 16

Competitor cell : target cell ratio

FIG. 5. Competitive ability of IFN-treated and 5-FU-treated target cells. NK effector cells were cultured with 51Cr-labeled K-562 cells, at effector to target ratio of 5O:l. Unlabeled K-562 (a) cells, IFN-treated K-562 cells (300 unitslml, O), 5-F&treated K-562 cells (100 ngiml, x), IFN + 5-FU-treated K-562 cells (A), Eskol cells (01, IFN-treated Eskol cells (+ ), 5-FU-treated Eskol cells (8) and IFN + 5-FU-treated Eskol cells (A) were assayed for their competitive ability at various competitor to target ratios. The experiments were done in triplicates for 4 hr when the spontaneous slCr release was 4-16% and SEM was ,1.3--4.7. The curves represent the data of one of three experiments using effector cells from three different donors.

110 REITER ET AL.

FIG. 6. 5FU increases the susceptibility of target cells to NKCF. Eskol cells were pretreated with or without (II) the presence of 5-FIJ (1 rig/ml, ilU; 100 rig/ml, El; 1 kg/ml, 0) for 18 hr, 51Cr-labeled, and assayed for their sensitivity to NKCF. The assay was done in triplicate and the SEM was 2.0-56. The histograms present data of one of two experiments.

NK cells, which are probably triggered by the target cell, and leads to a rearrangement of the NK cytoplas- mic granules toward the site of binding with the target cell. The next step is the release of some of the granules or their contents from the effector cell. The cytotoxic molecule (NK cytotoxic factor, NKCFf would then bind to the target cell, presumably at sites separated from those recognized by the NK receptor (25, 26). Since 5-FU increased the sensitivity of tumor cells to NK- CMC and abolished the protective effect induced by IFN against NK-CMC!, we addressed the question in which steps of the killing process 5-FU is involved.

J are also metabolized to FdUMP which inhibits thymidylate synthetase by formation of a covalent ter- nary complex. The consequent thymidine depletion inhibits DNA synthesis and may be synergistic with other antiproliferative agents, including IFN, simply by inhibiting the same pathway at different points. Fi- nally, numerous interactions due to altered metabolism of FU derivaties or cellular components as well as interactions based upon cell cycle perturbations or modulation of tumor subpopulations are possible (13 1,

Although the molecular mechanisms involved in the interaction of IFN-a and 5-FU are not yet identified, it is clear that this combination has great clinical potential. A major problem of chemotherapeutic and immu- notherapeutic treatment is severe side effects. Combi- nation of IFN and 5-FU may permit the reduction of the total dose of both drugs.

IFN is neither involved in the ability of tumor cells to form conjugates with the effector cells nor in the regulation of the sensitivity of tumor cells to NKCF (26). However, IFN reduces the ability of tumor cells to activate NK cells (following interaction with target cells) and reduces the induction of NKCF release from the effector cells. We have found that 5-FU does not affect the conjugation step nor the effector cell induction. However, our results indicate that 5FU enhanced the direct sensitivity of tumor cells to NKCF. Combination treatment of 5-FU and IFN resulted in increased tumor cell susceptibility to NK-CMC and/or abolished the protection against NK activity induced by IFN. These effects may reflect the enhancement of target cell sensitivity to NKCF induced by 5-FU. More studies are needed in order to examine the effect of 5-FU treatment on the sensitivity of tumor cells to other cytotoxic-immune mechanisms such as the cytotoxic T lymphocytes (CTL), tumor necrosis factor (TNF), antibody-dependent cell cytotoxicity (ADCC), and the complement system.

Based upon the known cellular pharmacology of IFN-a and 5FU, several tvnes of interaction are DOS- I

sible. One major pathway of 5-FU cytotoxicity is its incorporation into RNA, following metabolism to the nucleotide FUTP (13). Since IFN may also influence the rate of RNA metabolism, a synergistic effect could occur as a result of combined effects upon this pathway. Antagonistic interactions are also conceivable, based upon possible IFN inhibition of FUTP incorporation into RNA. It is alternatively possible that RNA which has inco~orated FU bases would not be as efficient as normal RNA in serving as a template for IFN-induced proteins necessary for IFN effects. Incorporation of FU derivatives, via the metabolite FdUTP, into DNA has also been demonstrated and this pathway could inter- act with IFN in similar ways. 5FU and its derivatives

In this paper, we report that IFN and 5FU have an in vitro additive antiproliferative effect on various tumor cells. We have also suggested that this combination may be involved in the in uiuo regulation of the NK system against tumor cells and possibly tumor me- tastasis. In order to confirm this hypothesis, more studies are needed in which NK cells from cancer patients will be examined for their activity following either in uiuo or in vitro treatments with IFN-(w and 5FU

REFERENCES

1. Borden, E., Effects of interferons in neoplastic diseases of man. Pharmac. Ther. 37, 213-229, 1988.

2. Gresser, I., Antitumor effects of interferon. Acta Oncot. i?8, 347- 353, 1989.

3. Murren, J. R., and Buzaid, A. C., The role of interferons in treatment of malignant neoplasms. Yale J. Biol. Med. 62, 271-290, 1989.

4. Okita, K., and Kaneko, T., The potential of interferons in malignant disease. Drugs 39, l-6, 1990.

5. Namba, M., Miyoshi, T.. Kanamori, T., Nobuhara, M., Kimoto, T., and Ogawa, S., Combined effects of 5-flurouracil and interferon on proliferation of human neoplastic cells in culture. Gann. 73, 819-824, 1982.

6. Miyoshi, T., Ogawa, S., Kanamori, T., Nobuhara, M., and Namba, M., Interferon potentiates cytotoxic effects of 5-ffuoro- __ _. uracil on cell proliferation of established human cell lmes orig- inating from neoplastic tissues. Cancer Let& 17, 239-247, 1988.


7. Yamamoto, S., Tanaka, H., Kanamori, T., Nobuhara, M., and Namba, M., In vitro studies of cytotoxic effects of anticancer drugs by interferon on a human neoplastic cell line (HeLa). Can- cer Lett. 20, 131-138, 1983.

8. Stolfi, R. L., and Martin, D. S., Modulation of chemotherapeutic drug activity with polyribonucleotides or with interferon. J. Biol. Response Modif. 4, 634-639, 1985.

9. Kimoto, Y., Antitumor effect of interferons with chemotherapeutic agents. Gun. to Kaguku, Ryoko. 13, 293-301.

10. Elias, L., and Crissman, H. A., Interferon effects upon the adenocarcinoma 38 and HL-60 cell lines: Antiproliferative re- sponses and synergistic interactions with halogenated pyrimi- dine antimetabolites. Cancer Res. 48, 48684873, 1988.

11. Walder, S., Schwartz, E. L., and Goldman, M., Flurouracil and recombinant alpha-2a-interferon: An active regimen against advanced colorectal carcinoma. J. C&n. Oncol. 7, 1769-1775,1989.

12. Pazdur, R., Abbruzzese, J., and Faintueh, J., Phase II study of recombinant interferon alpha (rIFN) and 5-flurouracil(5-FU) in patients with advanced colorectal carcinoma. PFOC. ASCO 9, 455-461,1990.

13. Elias, L., and Sandoval, J. M., Interferon effects upon flurouracil metabolism by HL-60 cells. Biochem. Biopkys. Res. Commun. 163, 867-874,1989.

14. Logothetis, C. J., Hossan, E., Sella, A., Dexeus, F. H., and Am- ato, R. J., Flurouraeil and recombinant human interferon alpha- 2a in the treatment of metastatic chemotherapy-refractory urothelial tumors. J. Nut. Cancer Inst. 83, 285-288, 1991.

15. Trinchieri, G., and Santoli, D., Antiviral activity induced by cul- turing lymphocytes with tumor derived or virus transformed cells. Enhancement of human natural killer cell activity by interferon and antagonistic inhibition of susceptibility of target cells to lysis. J. Z&p. Med. 147, 1314-1333, 1978.

16. Reiter, Z., Fischer, D. G., and Rubinstein, M., The protective effect of interferon against natural killing activity is not mediated via the expression of class I MHC antigens. Zmmunol. Lett. 17, 232-239, 1988.

Received June 20, 1991; accepted with revision September 20, 1991

17. Rosenberg, S. A., Lotze, M. T., Muul, L. M., Leitman, S., Chang, A. E., Vetto, J. T., Seipp, C. A,, and Simpson, C., A new approach to the therapy of cancer based on the systemic adminis- tration of autologous lymphokine-activated killer cells and recombinant interleukin-2. Surgery 109, 263-271, 1986.

18. Lahat, N., Aghai, E., Kotler, A., Kinarty, A., Sobel, E., Gruener, N., and Froom, P., Discordant effect of interferon on natural killer activity and tumor cell sensitivity to lysis in hairy cell leukemia. Blood 71, 1141-1143, 1988.

19. Harvey, W., Srour, E. F., Turner, R., Carey, R., Maze, R., Star- rett, B., Kanagala, R., Pereira, D., Merchant, P., Taylor, M., and Jansen, J., Characterization of a new cell line (ESKOL) resem- bling hairy cell leukemia: A model for oncogene regulation and late B cell differentiation. Leukemia Res., 15, 733-744, 1991.

20. Roth, W. K., Werner, S., Risau, W., Remberger, K., and Hof- schneider, P. H., Cultured, AIDS-related Kaposi’s sarcoma cells express endothelial cell markers and are weakly malignant in vitro. Znt. J. Cancer 42, 767-773, 1988.

21. Rager-Zisman, B., and Bloom, B., Interferons and natural killer cells. Brit. Med. Bull. 41, 22-27, 1985.

22. Ujiie, T., Increased sensitivity of tumor cells to immune defence cells following treatment with antineoplastic agents in vitro. Japan. J. Exp. Med. 59, 17-26, 1989.

23. Charamella, L. J., Meyer, C., Thompson, 0. E., and Dimitrov, N. V., Chemotherapeutic agents and modulation of natural killer cell activity in vitro. J. Zmmwwpkarm. 7, 53-65, 1985.

24. Harris, J. E., and Sinkovics, J. G., 1976. Suppression of human immune response. In “The I~unolo~ of Malignant Disease”. CV Mosby Co., St. Louis.

25. Trinchieri, G., The biology of natural killer (NK) cells. Adu. Zmmun. 47, 187-376,1989.

26. Wright, S. C., and Bonavida, B., Studies on the mechanism of natural killer mediated cytotoxicity. IV. Interferon-induced inhibition of NK target cell susceptibility to lysis is due to a defect in their ability to stimulate release of natural killer cytotoxic factor (NKCF). J. Zmmunol. 130, 2965-2968, 1983.

A dual anti-tumor effect of a combination of interferon-α or interleukin-2 and 5-fluorouracil on...

Documents

Transcript of A dual anti-tumor effect of a combination of interferon-α or interleukin-2 and 5-fluorouracil on...