Europ. J. Cancer Vol. 12, pp. 859-864. Pergamon Press 1976. Printed in Great Britain

In Vitro Inhibition of Cell Growth

by Aldose Phosphates* ALLAN FENSELAUt~ and CEDRIC LONG§ ~Department of Physiological Chemistry, The John Hopkins University School of Medicine, Baltimore, Maryland 21205, and §Flow Laboratories, Inc., Rockville, Maryland 20852, U.S.A. Abstract--The ~ c t s of various aldehydes and phosphoaldehydes on cell growth were examined using normal 3 T3 mouse fibroblasts and 3 T3 mouse cells transformed with 8V40, polyoma and Kirsten sarcoma viruses. I~L-Glyceraldehyde-3-phosphate, Derythrose-4-phosphate, and D-ribose-5-phosphate (all at less than 1 mM) inhibited the growth of actively dividing 3 T3 mouse cells more than their corresponding non-phosphorylated aldoses; only glycolaldehyde was more active than its phosphate. Addition of glutathione (0.5 m~f) potentiated these effectsfor the phosphoaldehydes, but generally had little influence or•! the effectsproduced by the neutral aldehydes. The growth of transformed cells in the log phase was inhibited by glyceraldehyde phosphate to about the same extent: however, in a semi-resting (confluent) state, the transformed cell lines were significantly less affected by glyceraldehyde phosphate than the normal cell line. Although a mechanismfor this action cannot be proposed, the conversion ofglyceraldehyde phosphate into two known inhibitors of cell growth, z-glyceraldehyde and methylglyoxal, does not seem to accountfor its potency and the thiol effect.

neutral aldehydes on the growth of normal and virally-transformed 3T3 mouse cells [20-22].

INTRODUCTION VARIOUS aldehydes and ketoaldehydes can inhibit in vivo and in vitro mammalian cell growth by interfiMng with intermediary metabolism, protein synthesis or possibly other cellular processes [1-17]. Several of these substances, most notably kethoxal [9] and glyceraldehyde [10, 18], offer some promise as chemotherapeutic agents in the treatment of solid tumors and leukemia. The aldehydes studied to date can freely penetrate into the cell, since they mainly lack charged groups that can limit their passage through the plasma membrane [19]. On the other hand, phosphoaldehydes as such should be unable to enter cells, but their inhibitory effects have not been examined. In the following we would like to report the results of our in vitro studies on the effects of various phosphoaldehydes, particularly I)L-glyceraldehyde-phosphate, as well as

MATERIAL AND METHODS The cell lines employed in these studies were the random bred Swiss mouse 3T3, polyoma virus-transformed 3T3 (Py 3T3), SV 40 virustransformed 3T3 (SV 3T3), all kindly provided by Dr. H. Green [21, 22], and Kirsten sarcoma virus-transformed Balb/c mouse embryo fibroblasts (KiSV), generously supplied by Dr. S. Aaronson [20]. The cells were grown in Eagle's minimal essential medium (EMEM) (Flow Laboratories, Rockville, Md.) supplemented with 10 % calf serum or 5 % heated calf serum (Colorado Serum Co. Laboratories, Denver, Colo.), glutamine, and antibiotics. The effects of various agents on cell growth were determined on cells that were actively dividing (log phase) or on cells in a semi-resting state (confluence). For most of the log phase studies, trypsinized cells (1.5x 10 5) were plated in plastic dishes (Falcon, 6 . 0 c m diam.) and allowed to grow 24 hr on the usual growth medium. Then the medium was replaced with fresh, sterile medium containing the drug. The cells were observed periodically over the

Accepted 9 June 1976. *This work was supported by grants from the National Science Foundation (GB 20672) and National Cancer Institute of the USPHS (CA 11699) and by a contract with the Virus Cancer Program of the National Cancer Institute. ++Research Career Development Awardee, USPHS, National Institute of General Medical Sciences (GM 70423). 859

860

Allan Fenselau and Cedric Long

next two days and were counted 72 hr after drug addition using standard procedures (Trypan blue dye exclusion). In the case of cell density dependency studies, initial concentrations of approximately 2 x 105, 5 x 105 and 10 x 10 s cells were plated, 6 hr later media were changed, and after an additional 18-20 hr cell counts were obtained. For studies on cells in the semi-resting state, 106 cells were plated in a dish and allowed to approach confluence in E M E M with 10 % calf serum (usually by over 3-4 days), after which fresh medium containing 5 % heated calf serum was added. Under these latter conditions normal mouse fibroblasts became confluent and stopped dividing, while cells transformed by SV 40, polyoma or sarcoma viruses continued to divide. After 24 hr, fresh medium with the drug included was added; the plates were observed over 72hr and the cells were counted at the end of this period. Duplicate plates were employed in all experiments. Results are expressed in terms of the ratio R of the treated-cell counts to untreated (control) cell counts. For R = 0 no viable cells were counted following drug treatment. DL-Glyceraldehyde-3-phosphate (Sigma Chemical Corp., St. Louis, Mo.), dihydroxyacetone phosphate (Sigma) and glycolaldehyde phosphate (Calbiochem, La Jolla, Cal.) were prepared from the ketals of the barium salts using Dowex-50 according to the supplier's instructions. Solutions containing ethanol and Ba(OH) 2 . 8 H 2 0 at concentrations equivalent to those from the preparation of the organic phosphates (as free acids) were treated in a similar fashion, but failed to display any effects in cell growth. A mixture of tetrose 2,4bisphosphates was synthesized from glycolaldehyde phosphate and purified according to the published procedure [23]. Glutathione was purchased from Schwartz-Mann (Orangeburg, N.Y. ; lot no. V3705). All other chemicals were obtained from various commercial sources and were the highest purity available. RESULTS A survey of the effect of various substances was made by using log phase cultures of the 3T3 mouse cell line. In Table 1 may be seen the results of one such study using several phosphoaldehydes and the corresponding dephosphorylated analog. First, the observation that L-glyceraldehyde is a more effective growth inhibitor than the D-isomer has been noted previously [24] and is confirmed by our in vitro studies. However, the DL-phosphate is a much more potent inhibitor than L-glycer-

aldehyde at the same concentrations. Second, the phosphomonoesters in general are better inhibitors than the corresponding unphosphorylated derivatives. Finally, the aldose phosphates are much more active than another phosphoaldehyde, pyridoxal phosphate, which is commonly employed in preparing Schiff base derivatives with soluble proteins [25] and cell membrane components [19]. Table 1. Effects of various aldehydes and their phosphate derivatives on the growth of 3 T3 cells in the log phase The conditions employed in these studies are described under Material and Methods. Results are expressed in terms of the ratio (R) of treated cell counts to untreated cell counts.

Glycolaldehyde Glycolaldehyde phosphate

D-Glyceraldehyde

L-Glyceraldehyde

9,L-Glyceraldehyde-3-phosphate

D-Erythrose D-Erythrose-4-phosphate D-Ribose D-Ribose-5-phosphate Pyridoxal Pyridoxal phosphate

Clnc., mM

R

>1 0.2 0-6 0.4 0.2 0.6 0.4 0.2 0.6 0.4 0.2 0-6 0.4 0.2 0.8 0.4 0.8 o.4 0.8 0-4 0.8 0.4 0.8 0.8

0.0 0.17 0.21 0.67 0.67 1.05 1.05 0.51 0.70 0.86 0.23 0.47 0.54 0-82 0.94 0.11 0.72 1.00 1.06 0.68 0.78 1.02 0.98

From the list of inactive compounds (Table 2) it appears that the presence of an aldehydic group alone (as in the aliphatic aldehydes) or a phosphate group alone in the test substance is insufficient for producing an inhibition of in vitro cell growth. Reduction or oxidation of the aldehyde in glyceraldehyde phosphate to give fl-glycerophosphate or 3phosphoglycerate, for example, eliminates activity. Isomerization of the carbonyl group to provide dihydroxyacetone phosphate also abolishes most of the activity. The more potent drugs (Tables 1 and 2) contain an aldehydic group with an adjacent

In Vitro Inhibition of Cell Growth by Aldose Phosphates oxygen (or sulfur) functional group on a short chain of carbon atoms. A phosphate group need not be present for the display of growth retardation or cell killing. Among the active compounds diffi:rences in inhibitory powers Table 2. Structural analogs of glyceraldehyde phosphate with little or no effect on the growth of 3 T3 cells in the log phase The conditions employed in these studies are described under Material and Methods. Addition of glutathione (0.5 raM) did not lead to a potentiation of an inhibition by any of the following compounds. The ratio of treated cell counts to untreated cell counts varied between 0.85 and 1.0 for the fMlowing.The highest concentration (raM) of the analog employed is given in parentheses. Glycollate (0.4) Glyoxalate (0.4) Pyruvate (0.4) Lactate (0.4) Acetaldehyde (0-4) Propionaldehyde (0.6) Hydroxyacetone (0' 7)

3-Phosphoglycerate (0.7) p-Glycerophosphate (0.7) e-Glycerophosphate (0-7) Acetylphosphate (0.4) Phosphoenolpyruvate (0-4) ATP (0.4) Dihydroxyacetone phosphate (0"6)

Dihydroxyacetone(0.7) Fructose- 1,6-bisphosphate

(0.5)

861

methylglyoxal and a-mercaptoglyceraldehyde in Table 3) ; a potentiation of the inhibition was observed, nevertheless, in the case of glyoxal (from 0.2-0.8 mM). However, all of the aldose phosphates examined exhibited enhanced inhibition in combination with glutathione (Table 3). The addition of glutathione to solutions of the corresponding aldoses did not significantly alter their inhibitory properties. The possibility that the synergistic effects with glyceraldehyde phosphate might be due to the formation of a polymeric, polyanionic inhibitor (containing thioacetal linkages between the aldehyde and thiol molecules) was tested using heparin ( < 200 #g/ml), but no growth inhibition by the polyanion was observed. Table 3. Effects of various aldehydes in the presence and absence of glutathione on the growth of 3 T3 cells in the log phase Conditions employed in these studies are described under Material and Methods. Results are expressed in terms of the ratio (R) of treated cell counts to untreated cell counts and are the average of two separate experiments. The concentration (raM) of the added agent is given in parentheses.

2-Deoxyribose (0-6) R

were noted; specifically, the glyceraldehydes, glyoxal, methylglyoxal (pyruvaldehyde), and a-mercaptoglyceraldehyde were not as effective over a concentration range of 0.2-0.8 m M in inhibiting cell growth as kethoxal, glycolaldehyde, DL-glyceraldehyde phosphate, and glycolaldehyde pl)osphate. The effects of added thiols, which can partially reverse the inhibitions of the neutral aldehydes [7, 13], were also examined in our in vitro system. With dithiothreitol, dithioerythritol, cysteine, N-acetyl-cysteine, flmercaptoethanol:, and aminoethanol (and in the absence of ai[dehydes), significant retardation of growth of~:he normal 3T3 cells was noted in all cases when calf serum was used [the ratio (R) of treated cell counts to untreated cell counts = 0.5-0.75 at 1.2 m M thiol]. However, significantly less of an effect (R=0.75-1.0) resulted when fetal calf serum was substituted. Most important in this regard is that the inhibition by DL-glyceraldehyde phosphate alone is independent of serum. The addition of glutathione (0.5 raM) to media containing the various test substances (at several concentrations from 0.2-0.8 mM) had only a slight effect on the inhibition produced by neutral aldehydes. Generally the result was a small reversal of the inhibition (as shown by kethoxal,

R

No Glutathione glutathione (0.5 mM) added added DL-Glyceraldehyde-3-phosphate (O.4) Glycolaldehyde phosphate (0.1) Threose-2,4-bisphosphate (0.3) D-Erythrose-4-phosphate (0.4) D-Ribose-5-phosphate (0.8) Methylglyoxal (0.6) Glyoxal (0.4) e-Mereaptoglyceraldehyde (0.6) Kethoxal (0.3)

0.48

0.1 t

0.68 0.70 0.68 0.61 0.45 0.86 0.54 0.07

0.18 0.41 0.34 0.38 0.56 0.55 0.53 0.20

The inhibition studies were extended to virally-transformed 3T3 cells in both the growing (log phase) and semi-resting (confluent) states, using only the more readily available DL-glyceraldehyde phosphate. The four cell lines, the normal 3T3 and the Py, SV40, and KiSV transformed derivatives, were subcultured at various initial cell population densities (8.5, 17 and 34 x 103 cells/cm 2) and were treated with varying amounts of DL-glyceraldehyde phosphate (0.45-1-35 m M at the lowest initial densities and 1-3 m M at the highest). At low initial cell densities, only the KiSV 3T3 cultures contained significant cell numbers (R = 0.40) when high concentrations of glyceraldehyde phosphate were used;

862

Allan Fenselau and Cedric Long

at lower concentrations, both KiSV 3T3 and SV 3T3 cultures had higher R values (0.70 and 0.33, respectively). This lesser sensitivity shown by two of the transformed cell lines toward glyceraldehyde phosphate inhibition was more evident at the higher initial cell densities. For example, at 34 x 10 3 c e l l s / c m 2 and 1 m M glyceraldehyde phosphate essentially no growth retardation was observed with the three transformed cell lines, whereas R=0.22 for the normal 3T3 cell line; at 3 m M glyceraldehyde phosphate, though, only the KiSV 3T3 culture had a high R value (0.55). A similar result was found when the different cell lines were grown to confluence in a medium containing 10 % serum, were then adapted to a semi-resting state in a maintenance medium containing heat-inactivated serum, and finally were overlaid with a maintenance medium containing glyceraldehyde phosphate (1-3 raM). At 1 m M aldose phosphate R---0.28 for the 3T3 cells, but for the transformed cells the R-values ranged from 0.73-0.88. Growth of both Py 3T3 and KiSV 3T3 cell lines was inhibited significantly at 3 m M glyceraldehyde phosphate (R=0-43-0.46); however, no cells were counted for normal 3T3 cultures that were treated identically. DISCUSSION

The results of these studies indicate that aldose phosphates, especially glyceraldehyde phosphate and glycolaldehyde phosphate, are inhibitors of 3T3 cell growth in vitro. This inhibition is greater than that found in vitro with the corresponding neutral aldoses (with glycolaldehyde as the lone exception) and comparable to that found with several substances (L-glyceraldehyde, kethoxal and methylglyoxal), which have undergone limited testing as possible cancer chematherapeutic agents [1, 9, 18]. The potency of the phosphoaldehydes is enhanced further by the addition of a thiol compound, such as glutathione. Inhibition by glyceraldehyde phosphate, and presumably by the other phosphoaldehydes, is greatest with sparse cultures of actively dividing cells, which can be either normal or virallytransformed. However, the resistance of the transformants to the effects of glyceraldehyde phosphate, barely noticeable with sparse cultures, becomes more evident in confluent (or serum-deprived) cultures. No explanation can

be advanced at this time for these results, which certainly merit further study. The site of action for these phosphorylated aldehydes is not at all clear. The relative impermeability of the mammalian membrane to organophosphates [19] and the greater activity in vitro of the charged versus the corresponding uncharged aldehydes suggest that the aldose phosphates may be acting at different or additional loci. Inhibition by the neutral glyceraldehydes occurs with the L-form at the hexokinase step in glycolysis (due to the formation of the active species sorbose phosphate by aldolase [24]) and in protein synthesis with both D- and L-forms [11, 12]. It also appears that the inhibition by the aldose phosphate involves more than formation of Schiff base derivatives with soluble serum proteins or cell membrane components, since the aldose phosphates are more active than pyridoxal phosphate. Finally, the identity of the active species for these inhibitory effects also remains to be established. Culture media composed of amino acids in high concentration and serum containing many undefined proteins, along with the thiol, might very well chemically alter aldehydes or phosphoaldehydes. In fact the intracellular formation of thiazolidine-4-carboxylic acids from added aliphatic aldehydes (which readily enter mammalian cells) and endogenous cysteine has been invoked as an explanation for the in vivo inhibitory effects of the aldehydes on protein biosynthesis [13]. Furthermore, the conversion to methylglyoxal from glyceraldehyde [26] or glyceraldehyde-3-phosphate [27] can be non-enzymically catalysed by lysine, although methylglyoxal formation does not seem to be stoichiometrically related to (phospho)aldehyde loss. Our own results indicate that glyceraldehyde phosphate alone is more active against normal 3T3 cells than glyceraldehyde or methylglyoxal over a range of concentrations (0.2-0.8mM) and that this growth inhibition by the phosphoaldehyde is augmented by the presence of added extracellular glutathione (but is neither reversed nor unaffected, as with the neutral aldehydes). More detailed study, however, is still needed in order to determine the mechanism(s) of action for the inhibitions by aldose phosphates. Acknowledgements--We gratefully acknowledge the skilled technical assistance of Mrs. Tena Wei and Mr. Bob Baker.

In Vitro Inhibition of Gell Growth by Aldose Phosphates

REFERENCES 1.

2vl. A. APPLE and M. J. CLINE,Anticancer effects of propanals: comparison between human and animal tumor systems. Nat. Cancer Inst. Monogr. 34, 161

(1971).

2.

lvI. A. APPLE and D. M. GREENBERG,Arrest of cancer in mice by therapy with normal metabolites. 1. 2-oxopropanal. Cancer Chemother. Rep. 51, 455 (11967). 3. M . A . APPLE and D. M. GREENBERO, Arrest of cancer in mice by therapy with normal metabolites. II. Indefinite survivors among mice treated with mixtures of 2-oxopropanal and 2,3-dihidroxypropanal. Cancer Chemother. Rep. 52, 687 (1968). 4. M . A . APPLE and D. M. GREENBERO, Inhibitory effect of DL-2-mercapto-3tlydroxypropanal on growth of transplantable cancers in mice. CancerChemother. Rep. 53, 195 (1969). 5. ~VI.i . APPLE, F. C. LUDWIGand D. M. GREENBERO,Selective growth inhibition in mice by dihydroxypropanal without concomitant inhibition of bone marrow or other normal tissue. Oncology 249 210 (1970). 6. E. CIARANFI,L. LORETI,i . BORGHETTIand G. G. GUIDOTTI,Studies on the anti-tumor activity of aliphatic aldehydes. II. Effects on survival of Yoshida ascites hepatoma-bearing rats. Europ. J. Cancer 1~ 147 (1965). 7. L . G . EGYOD, Studies on cell division: the antagonism of ~-ketoaldehydes and thiols. Curt. Mod. Biol. 2~ 128 (1968). 8. 13. FREEDLANDERand F. A. FRENCH,Carcinostatic action of polycarbonyl compounds and their derivatives. II. Glyoxal bis (guanylhydrazone) and derivatives. Cancer Res. 18~ 360 (1958). 9. F.A. FRENCH and B. L. FREEDLANDER,Carcinostatic action of polycarbonyl compounds and their derivatives. I. 3-ethoxy-2-ketobutyraldehyde and related compounds. Cancer Res. 18~ 172 (1958). 10. B.C. GIOVANNELLA,W. A. LOHMANand C. HEIDELBERGER,Effects of elevated temperatures and drugs on the viability of L1210 leukemia cells. CancerRes. 30, 1623 (1970). 11. G. G. GUIDOTTI, A. FONNESU and E. CIARANFI, Inhibition of amino acid incorporation into protein ofYoshida ascites hepatoma ceils by glyceraldehyde. Cancer Res. 24~ 900 (1964). 12. G.G. GUIDOTTI,L. LORETIand E. CIARANFI,Studies on the antl-tumor activity ofaliphatic aldehydes. 1. The mechanism of inhibition of amino acid incorporation into protein ofYoshida ascites hepatoma cells. Europ. J. Cancer1, 23 (1965). 13. L. LORETI, M. E. FERIOLI, G. C. GAZZOLOand G. G. GUIDOTTI, Studies on the anti-tumor activity of aliphatic aldehydes. III. Formation of thiazolidine4-carboxylic acid in tissues. Europ. J. Cancer 7, 281 (1971). 14. E. MIHICH and C. A. NICHOL, Kethoxal bis(thiosemicarbazone). I. Effects ~gainst experimental tumors. Cancer Res. 2fi~ 1410 (1965). 15. A. SAKAMOTOand K. N. PRASAD, Effect of I)L-glyceraldehyde on mouse neuroblastoma cells in culture. Cancer Res. 32, 532 (1972). 16. A. C. SARTORELLIand W. A. CREASEY, Cancer chemotherapy. Ann. Rev. Pharmacol. 9~ 51 (1969). 17. A. SZENT-GYORGYI,L. G. EOYf3D and J. A. McLAuGHLIN, Keto-aldehydes a.nd cell division. Science 155, 539 (1967). 18. }¢I. YON ARDENNE, Zur Berechnung der Glycerinaldehyd-Wirkdosis im Gewebe bei Applikation in den Kreislauf. Naturwissenschaften $1, 217 (1964). 19. D.B. RIFKIN, R. W. COMPANSand E. REICH, A specific labeling procedure for proteins on the outer surface of membranes, or. biol. Chem. 247~ 6432 (1972). 20. J. R. STEPHENSON and S. A. AARONSON, Antigenic properties of murine sarcoma virus-transformed BALB/3T3 non-producer cells, d. expt. Med. 135~ 503 (1972). 21. G . J . TODARO and H. GREEN, Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines, d. Cell. Biol. 17, 299 (1963). 22. G.J. TODARO, H. GREEN and B. D. GOLDBERG,Transformation of properties of an established cell line by SV40 and polyoma virus. Proc. nat. Acad. Sci. (Wash.) 51, 66 (1964).

863

864

Allan Fenselau and Cedric Long 23. 24. 25. 26. 27.

A . L . FLUHARTYand C. E. BALLOU,D-Threose-2,4-diphosphate inhibition of D-glyceraldehyde-3-phosphate dehydrogenase. J. biol. Chem. 234, 2517 (1959). H.A. LARDY,V. D. VIEBELHAUSand K. M. MANN, The mechanism by which glyceraldehyde inhibits glycolysis. J. biol. Chem. 187, 325 (1950). G.E. MEANSand R. E. FEENEY,Chemical Modification of Proteins Holden-Day, Inc., San Francisco (1971). A. BONSIGNORE,G. LEONCINI,A. SIRI and D. RiceI, Kinetic behaviour of glyceraldehyde-3-phosphate conversion into methylglyoxal. Ital. J. Biochem. 22, 131 (1973). V. RIDDLE and F. W. LORENZ, Non-enzymic, polyvalent anion-catalyzed formation of methylglyoxal as an explanation of its presence in physiological systems. J. biol. Chem. 2't3, 2718 (1968).

In vitro inhibition of cell growth by aldose phosphates.

Europ. J. Cancer Vol. 12, pp. 859-864. Pergamon Press 1976. Printed in Great Britain In Vitro Inhibition of Cell Growth by Aldose Phosphates* ALLAN...
NAN Sizes 0 Downloads 0 Views