273
EFFECTOF DISULFIDES ASD THIOXES o x , \ S C I T E ~ Tuiron
>larch 1970
The Effect of Some Heterocyclic Disulfides and Thiones on the Carbohydrate Metabolism of Ehrlich Ascites Tumor’ D. R.GRASSETTI, J. F. ;\IURRAT, ,JR., 11. E. BROKKE, ASD -1.D. GUTMAA dreqitzpa Foundation, Sun Fiancisco, Calijorniu 94104
Receiced September 19, 1969 The effect of six thione-forming disulfides and five corresponding thioiiea 011 carbohydrate metabolism of Ehrlich ascites tumor has been studied by manomet,ric and isotopic methods. The effects observed confirm previous observations in that basic disulfides are strong inhibitors of glycolysis and respiration, while acidic disulfides, as well as most of the thiones, have little or no effect. T a o thiones however affect Carbohydrate metabolism. 5-Cyano-(2-t,hiopyridone) appears to prevent the aerobic utilization of exogenous glucose, whereas benzothiazole-2-thione causes a partial reversal of the Crabtree effect.
We have recently studied the effect of some thioneforming disulfides on the carbohydrate metabolism of Ehrlich ascites tumor cell^,*^^ and their interaction with Ehrlich ascites cells and h o m o g e n a t e ~ . ~ -In ~ our continuing study of the effect of disulfides and thiols on cell metabolism, i y e are now presenting the results obtained with qeveral thione-forming disulfides arid the corresponding thiones. These disulfides have the property of reacting with thiols in an essentially complete and irreverhible manner, as exemplified by 2,2’-dithiobis(isonicotinic acid). The products are a disulfide and a heterocyclic thiorie (in this case 4carboxy-2-thiopyridone). The thione nature of these compounds is evidenced by their uv ~ p e c t r a . ~ Materials and Methods.-Swiss mice, bearing 5- to 10-day old ascites tumor, were sacrificed by cervical fracture. The fluid n as collected, pooled, heparinized (50 USP units/ml), and used immediately. Manometric determinations 11ere carried out in a conventional Karburg apparatus a t 3 i 0 . Readings u ere taken a t 5 m i n intervals for 1 hr or more after introduction of the compound under study. The detailed procedure for the manometric experiments, as well as the determination of solubilities, was given previously.* The isotopic experiments were performed as described elsewhere;6 the experiments with IV and V were performed three times, those with VI11 twice, all with closely agreeing results. The result of one individual experiment with each compound is reported in Figures 1, 2, arid 3. Results
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0 15 30 45 60 Each disulfide used (Table I) reacted quantitatively Minutes with cysteine to give the corresponding t h i ~ n e . ~ ~ ~ ~ ? ~ ~ The thiones were stable to air oxidation with the Figure 1.-Effect of .i-cyano-2-thiopyridoiie ( I T ) on aerobic production of I4CO?from [14C]glucoseby Ehrlich aacites tumor exception of the amino derivative 11, which was
(1) This investigation was supported by Public Health Service Research Grant CA-08538, from the National Cancer Institute. (2) D. R. Grasset.ti, R f . E. Brokke. and J. F. Murray, Jr.. J . .\fer/. Chem., 8 , 753 (1965). (3) D. R. Grassetti, J. F. Murray, ,Jr., Af. E. Brokke, and .1.D. Gutman. (bid., 10, 1170 (1967). (4) D. R. Grassetti and J. F. Murray, Jr., Arch. Biochem. Btophys., 119, 4 1 (1967). (5) D. R . Grassetti, J. F. Murray, J r . , and H. T. Ruan, Biochem. Pharmacol., 18, 603 (1969). (6) D. R . Grassetti and J. F. hlurray, Jr., ibid., 17, 2281 (1968). (7) D. R . Grassetti and J. F. -Murray, Jr., Anal. Chim. Acta, 46, 139
(1969). ( 8 ) T h e reaction of XI with thiols, which we have not reported previously, leads also quantitatively t o the thione V.
cells. Each flask contained 30 wmol of [L4C]glucose(1 pCi) and about 50 x lo6 washed Ehrlich ascites cells (about 13 mg dry weight) in Krebs-Ringer phosphate buffer ( p H 7.2). Total volume per flask was 3.0 ml. Incubations were carried out a t 37’. 14C02 wa> collected and counted a i reported previously:6 (0)I4CO2production by washed Ehrlich ascites cells; (A) “C02 production in the presence of 3 @mol of IT’. ( A ) Evolution of 14CO2 from 1-[14C]glucose (02-CO2, 95: 3). The figures plotted were obtained by subtracting the values for 1 4 C Orecovery ~ using 6-[14C]glucosefrom those obtained using l-[l*C]glucose. ( B ) Evolution of I4COrfrom 6-[14C]glucose(0,-COI, 95 : 3).
readily oxidized to the disulfide,? and could therefore not be studied under the present experimental condi-
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EFFECTOF DISULF~DES AND THIONES ON ASCITESTLXOR
March 1970
275
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Figure ?.-Effect of benzothiazole-2-thione (V) on aerobic production of l4C0?from [14C]glucose by Ehrlich ascites tumor 01 1 cells. Pee Figure 1 for experimental conditions. (0) 14C02 pro0 15 30 45 60 duction by washed Ehrlich ascites cells; (A) 14C02 production Minutes in the presence of 3.0 pmol of V. (A) Evolution of 1 4 C Ofrom ~ Figure 3.-Effect of ?J'-dithiobisi 3-aminopyridine) (VIII) 1-[1T]gliico.e (02-COS,95:5). The figures plotted were obtained on aerobic production of 14c'0, from [14C]glucose by Ehrlich by subtracting the values for I4CO2 recovery using 6-[14C](B) EVO~U- ascites tumor cells. See Figure 1 for experimental conditions. glucose f r o m those obtained using l-[14C]gl~~cose. (0) l4COZ production by washed Ehrlich ascites cells; (A) I4CO2 tion of l'C'C).! from 6. [ llC]glncose (OS-CO?,95:3). production in the presence of 1.5 iiniol of VIII. (4)Evolution of W O n from l-[14C]glucose(O*-CO,, 95: 5 ) . The figures plotted were obtained by subtract,iiig the values for I4CO2recovery using 6-[14C]glucose from those obtained using l-[14C]glucose. (B) tion-. Re-ult- of the manometric experiment- are Evolution of l4c0? from 6-[14C]gluc~~se (&COS, 95.5).
reported in Table I. Thiones.-IThereas thiones I, 111, and V I have marginal effects, IV and T' elicited a significant metabolic re3pon.e. Compound IV caused a stimulation of Qo. (G), and had no effect on the other properties studied (Table I). This stimulation corresponds to an apparent reversal of the Crabtree effect amounting to about TOYc. Further studies with l-[14C]glucose nnd 6- [14C]glucoseas substrates were undertaken. The aerobic nietabolism of glucose by Ehrlich ascites cells via the hexose monophosphate (HAIP) pathway in the pre+ence of IY, as judged from the production of I4COr from 1-[14C]glucose,after an initial stimulation, soon comes to a standstill (Figure 1-4). The conversion of the C-6 of glucose to CO, can be used as a measure of oxidation via the Krebs cycle. Results presented in Figure 1B suggest that IT' exerts a n inhibitory activity on this cycle. A more detailed study of the metabolic effects of this interesting compound is in progress and will be reported elsewhere. Thione V, while stimulating Qo, (G), also stimulates aerobic and anaerobic glycolysis. The stimulation of Qo (G) correaponds to an apparent reversal of the
Crabtree effect of about 5 0 5 , a. measured manometrically. Results of experiment. u.;ing the I4C-labeled glucoses (Figure 2 ) show that \- cauqed a slow inhibition of the HMP pathway under these conditions (Figure 2A), and a moderate .timulation of the Krebs cycle (Figure 3B). Thu., the obqerved manometric stimulation of Qo2 (G) i- confirmed by the isotopic experiments and appear. t o be due to a partial reversal of the Crabtree effect. Disulfides.-Disulfide5 T-I1 and S I have little effect on the manometric characteri-tics of Ehrlich ascites cells. Whereas this lack of effect may be due to the relative insolubility of compound S I . this is not the case of compound T'II. Compound VI11 inhibits Qo and glycolysis, and causes a 30-50y0 stimulation of Qo2 (G), which is follon-ed by inhibition after 1 hr. Isotopic experiments carried out under aerobic conditions (Figure 3) showed that VI11 causes a large .timulation of the HMP pathway (Figure 3A), and a moderate inhibition of (9) H G Crabtree, Btochem J , 23 536
1525).
