Biotherapy 5: 285-290, 1992. © 1992 KluwerAcademic Publishers. Printed in the Netherlands.

Effect of pyridoxine on tumor necrosis factor activities in vitro Eva Hofsli & Anders Waage Institute of Cancer Research, University of Trondheim, N-7006 Trondheirn, Norway Received 26 February 1992; accepted 24 July 1992 Key words:

pyridoxine, tumor necrosis factor

Abstract

Clinical trials with tumor necrosis factor (TNF) as an antitumor agent have so far given rather disappointing results. In this study we show that the naturally occuring vitamin B 6 compound, pyridoxine, enhances TNF-induced cytolysis of three subclones of a mouse fibrosarcoma cell line ( W E H I 164). The degree of pyridoxine-induced enhancement of TNF cytotoxicity seems to be dependent on the cells sensitivity to TNF, as the enhancement was much more pronounced in the relatively TNF resistant subclone act-R(cl.12)-WEHI 164, than in the very TNF sensitive subclone W E H I 164 clone 13. Furthermore, our study shows that pyridoxine, in contrast to its enhancing effect on TNF-induced cytotoxicity, rather inhibits TNF-induced growth of human FS-4 fibroblasts. Pyridoxine also enhances lymphotoxin (LT)-induced tumor cell killing and inhibits LT-induced fibroblast growth. Pyridoxine is a relatively non-toxic agent in vivo. Our results suggest that a combination of TNF and pyridoxine may be more efficient than TNF alone, in the treatment of cancer patients. Abbreviations: LT: lymphotoxin; MTT: (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide); TNF: tumor necrosis factor

Introduction

Tumor necrosis factor (TNF) (also called TNF-o~ or cachectin) was originally thought to have biological functions restricted to killing of tumor cells in vitro and in vivo [1]. It is now known that this predominantly monocyte-produced protein may induce a wide range of biological effects [2, 3], among which is growth stimulation of human fibroblasts [4,5]. Furthermore, several studies indicate that TNF may be a mediator in septic shock [6, 7] and inflammation [8], and that it is a central regulator of immunity [9, 10]. Pyridoxine is a naturally occuring vitamin B 6 compound which in the organism is converted to the physiologically active pyridoxal phosphate (PLP) [11, 12]. PLP acts as a coenzyme in numerous enzymatic reactions, including important reactions involved in the metabolism of aminoacids and carbohydrates, as well as in the

synthesis of proteins, neurotransmittors, and heme [11, 12]. In addition to its classical cofactor function, PLP may in vivo modify the actions of steroid hormones by means of interactions with the steroid receptor complex [13, 14]. In vitro, PLP inhibits platelet aggregation [12], and stimulates the synthesis of PGE 2 in rabbit kidney medulla slices [15]. While studying the effect of TNF on tumor cells resistant to several chemotherapeutic drugs (multidrug resistant cells) [16], we discovered that pyridoxine influenced the cytotoxic effect of TNF. The interaction with TNF activity is a new and potentially important effect of pyridoxine. Thus, the present study was undertaken to examine the extent and character of the pyridoxine effect on TNF-induced cytotoxicity, and whether pyridoxine influences other TNF activities. The results demonstrate that pyridoxine enhances the cytotoxic effect of TNF on subclones of a mouse

286 fibrosarcoma cell line, whereas it inhibits TNFinduced growth of human fibroblasts.

Materials

and methods

Reagents. Recombinant human TNF (rTNF) was kindly provided by Knoll/BASF, Ludwigshafen, FRG. Recombinant human lymphotoxin (rLT) was produced by Genentech, Inc., South San Francisco, CA, USA, and kindly supplied by Dr. G.R. Adolf, Boehringer Ingelheim, Vienna, Austria. Pyridoxine chloride was purchased from NAF-Laboratories A/S, Oslo, Norway. Methylparaoxybenzoate was obtained from Norwegian Medicinal Depot, Oslo, Norway. Cells. The murine fibrosarcoma cell line WEHI 164 parental (termed P-WEHI 164) was obtained from Dr. H.W. L0ms Ziegler-Heitbrock, Institute of Immunology, University of Munich, F.R.G. The multidrug resistant (MDR) WEHI 164 clone 12 cell line (termed act-R(cl. 12)-WEHI 164) was derived from P-WEHI 164 cells as previously described [17]. This M D R clone is approximately 100-fold less sensitive to the cytotoxic action of rTNF than the parental cell line (data not shown). Human diploid FS-4 fibroblasts [18] were kindly provided by Dr. J. Vilcek, Department of Microbiology, New York School of Medicine, NY. All cell lines were maintained in complete medium consisting of RPMI 1640 (Gibco, Paisley, UK) containing 10% fetal calf serum, 2.7 mM L-glutamine and 40 I~g/ml gentamicin. Due to drift in TNF resistance, the clone 12 cells were used within two weeks of thawing frozen cell samples. Cytotoxicity assay. WEHI 164 parental and actR(cl.12)-WEHI 164 cells were seeded in 6 mm microculture wells (Costar 3596, Cambridge, MA, USA), 2 x 104 cells in 100 p,1 complete medium, and different concentrations of rTNF and/or pyridoxine were added at the times indicated. The final volume in each well was adjusted to 110 lxl. After 20 h of incubation at 37°C, 10 ~1 M T r (3(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide) [19] at a concentration of 5 mg/ml in phosphate-buffered saline, pH 7.2, were added. The cultures were incu-

bated for 4 h at 37°C, and thereafter 50 txl of the supernatant were removed and replaced by 100 ~xl 0.04 N HC1 in isopropanol. After the dark blue formazan had been dissolved, the optical density (OD) of each well was measured with a Dynatech MR 600 Microplate reader (Deukendorf, FRG), using a test wavelength of 570 nm and a reference wavelength of 630 nm. Percent dead cells were calculated as: 1-

OD in wells with pyridoxine/rTNF X 100 OD in control wells without pyridoxine/rTNF

Growth assay. FS-4 cells were seeded in microtiter plates (Coster 3596), 1 x 104 cells in 100 txl complete medium, and cultured for 24 h at 37°C before rTNF and/or pyridoxine were added as described above. The cells were then incubated for further 72 h before 1 ixCi (3H)thymidine was added. Four hs later, the cells were trypsinized (0.25% trypsin (Gibco) for 2 min at 37°C) and harvested with a Titertek multiple cell harvester, and radioactivity was determined by liquid scintillation counting. Results are given as percentage increase in (3H)thymidine incorporation, calculated as: E-C C

- -

x

100

where E = cpm in the experimental wells containing rTNF and/or pyridoxine, and C = cpm in the control wells with rTNF and/or pyridoxine.

Statistics. Results are given as mean--standard deviation of quadruplicate incubations from single experiments. One of at least five similar experiments is shown.

Results

Effect of pyridoxine on TNF-induced cytotoxicity. Pyridoxine enhanced in a dose dependent manner the cytotoxic effect of rTNF in both the P-WEHI 164 and the act-R(cI.12)-WEHI 164 cells (Figure 1). Pyridoxine alone at concentrations as high as 1000 Ixg/ml did not induce a cytotoxic response in neither the P-WEHI nor the act-R(cI.12)-WEHI 164 cells (Figure 1). Pyridoxine concentrations above 1000 txg/ml in-

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Fig. 1. Effect of pyridoxine on cytotoxicity of rTNF to P-WEHI (A) and act-R(clJ2)-WEHl (B) 164 cells. The cells were cultured in various concentrations of rTNF and 0 ( Q - Q ) , 500 ( O - - O ) , and 1000 ((3-(3) l~g/ml of pyridoxine° Cytotoxicity was measured by the MTT assay as described in Materials and Methods.

duced cell death in a dose dependent manner (data not shown). At a concentration of 50pg/ml of rTNF, 500 p~g/ml of pyridoxine increased toxicity from 11% to 15% in the P-WEHI 164 cells (Figure 1A). At higher rTNF concentrations, the enhancing effect of pyridoxine was more marked. At a rTNF concentration of 5000 pg/ml, toxicity was increased from 55% to 81% in the presence of 500 i~g/ml pyridoxine. The enhancing effect of pyridoxine was more marked in the relatively TNF resistant act-R(cl.12)-WEHI cells. In these cells (at a rTNF concentration of 5000pg/ml), 500 ~g/ml of pyridoxine increased rTNF toxicity from 9% to 72% (Figure 1B). Similar results were also obtained with the highly TNF sensitive cell line WEHI 164 clone 13 [19] (data not shown). Thus, the data show that pyridoxine enhances the cytotoxic effect of TNF on 3 different subclones of the WEHI 164 cells. The clones differ in their sensitivity to TNF, and the effect of pyridoxine is greatest in the most TNF resistant subclone. Experiments showed that there was no difference between adding pyridoxine 30-60 minutes prior to, concomitantly with, or 30-60 minutes after rTNF (data not shown). Lymphotoxin (LT, also called TNFq3) is a lymphocyte-produced cytokine which both struc-

turally and functionally is quite similar to TNF, and binds to the same receptor as TNF [2, 2022]. We studied the effect of pyridoxine on recombinant human LT (rLT)-induced cytotoxicity in the parental WEHI 164 and act-R(cl.12) cell lines (Figure 2). The results showed that pyridoxine increased rLT-induced cytotoxicity in both cell lines (Figure 2). As for TNF, the effect was much more pronounced in the act-R(cl.12) W E H I 164 cell line, which is about 100 times less sensitive to the cytotoxic effect of rLT than the P-WEHI 164 cell line.

Effect of pyridoxine on TNF-induced growth. In order to examine the effect of pyridoxine on other TNF activities, we studied rTNF-induced growth of normal human fibroblasts in the absence and presence of pyridoxine. As shown in Figure 3A, pyridoxine inhibited in a dose dependent manner rTNF-induced growth of FS-4 fibroblasts. Pyridoxine at a concentration of 500 p~g/ml, which alone did not influence the growth of the FS-4 cells, reduced TNF-induced growth (at a rTNF concentration of 100 ~g/ml) from 167% to 15% (Figure 3A). This was not due to an increased cell death caused by pyridoxine in the presence of rTNF, as no significant difference in dead cell counts was observed using trypan blue exclusion (data not shown).

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Fig. 2. Effect of pyridoxine on cytotoxicity of rLT to P-WEHI (A) and act-R(cl.12)-WEHI (B) 164 cells. The cells were cultured in various concentrations of rTNF and 0 ( O - O ) , 500 ( O - - O ) , and 1000 ( O - G ) p.g/ml of pyridoxine. Cytotoxicity was measured by the MTT assay as described in Materials and Methods.

We have earlier shown that LT stimulates the growth of FS-4 cells [21]. In the present study we found that pyridoxine inhibited rLT-stimulated growth of FS-4 cells in a dose dependent manner (Figure 3B). Pyridoxine at a concentration of 500 Ixg/ml, reduced LT-induced growth (at a rLT concentration of 100 ng/ml) from 178% to 49% (Figure 3B). Similar to the effect of pyridoxine on rTNF/rLT-induced cytotoxicity, there was no difference whether pyridoxine was added prior

to, concomitantly with (Figure 3), or after rTNF/ rLT. As the commercially available stock solution of pyridoxine chloride also contained the preserving agent methylparaoxy-benzoate, control experiments were performed in order to ensure that the observed effects were not due to methylparaoxybenzoate. We did not find any effect of methylparaoxybenzoate on rTNF-induced responses (data not shown).

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Fig. 3. Effect of pyridoxine on rTNF (A) and rLT (B)-induced growth of FS-4 fibroblasts. Fibroblasts were cultered in various concentrations of rTNF/LT and 0 ( O - O ) , 250 ( O - - O and 500 ((3-(3) p~g/rnl of pyridoxine. Fibroblast growth was measured with (3H)thymidine as described in MateriaLs and Methods.

289

Discussion The present study demonstrates that pyridoxine enhances the cytotoxic effect of TNF on three subclones of a mouse fibrosarcoma cell line, while it inhibits the growth-stimulatory activity of TNF in human FS-4 fibroblasts. The degree of pyridoxine-induced enhancement of TNF cytotoxicity seems to vary according to the TNF sensitivity of the tumor cells. The same effect of pyridoxine is seen on LTinduced responses; an enhancement of LTinduced cytolysis in tumor cells, and an inhibition of LT-induced fibroblast growth. The parallel effect of TNF and LT is not suprising, since these two cytokines have functional and structural similarities, and also bind to the same receptor [2,20-22]. Furthermore, we have earlier shown that interferon gamma has the same antagonistic effect on LT-induced fibrob|ast growth as it has on TNF-induced growth [21]. It seems unlikely that the effect pyridoxine exerts on TNF activities in vitro is due to an interaction with the TNF receptor, as there was no difference when pyridoxine was added prior to, together with, or after TNF. Clinical trials with TNF as an antitumor agent have so far given rather disappointing results [23-25]. The treatment has been complicated by serious side effects, and the antitumor effect has not been too convincing. The direct cytotoxic effect of TNF against tumor cells probably contributes to the antitumor effect of TNF in vivo, although other mechanisms (selective effect against tumor blood vessels and immunoregulatory effects) also seem to be involved. Pyridoxine has a low toxicity in vivo [11], and thus may be administered to man in relative high doses without inducing serious side effects. Our results, showing that pyridoxine enhances the cytotoxic effect of TNF, suggest that a combination of TNF and pyridoxine may be more efficient than TNF alone in the treatment of cancer patients.

Acknowledgments The technical assistance of M. S~rensen and B. Stordal is gratefully acknowledged. The current

work was supported by grants from the Norwegian Cancer Society (Den Norske Kreftforening).

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Address for correspondence: Eva Hofsli, Department of Oncology, Trondheim Regional Hospital, N-7006 Trondheim, Norway