Effect of procaine on cultivated human WI-38 fibroblasts

Experimental Gerontology, Vol. 23, pp. 87-96, 1988 Printed in the USA. All rights reserved.

EFFECT

OF PROCAINE

0531-5565/88$3.00 + .00 Copyright¢ 1988PergamonPress plc

ON CULTIVATED

HUMAN

WI-38

FIBROBLASTS

E. PIGEOLET, M. RAES, A. HOUBION and J. REMACLE Laboratoire de Biochime Cellulaire, Facult6s Universitaires, Rue de Bruxelles, 5000 Namur, Belgium Abstract - - Procaine is a local anesthetic, also used in experimental gerontology and has been tested in cultivated human WI-38 fibroblasts. This molecule was found to enhance growth rate and cell densities in actively dividing cultures. As the cells aged, however, this stimulatory effect diminished and finally vanished. In a long term experiment the enhancement of growth of procaine treated cultures was finally replaced by a toxic effect even at low concentration. The amount of the thermolabile enzyme found in phase IIl cells did not change when procaine was added to the culture medium. In this cellular aging model, procaine behaved like a metabolic stimulator of actively dividing cells but not as an "antiaging" molecule as it is sometimes assumed. Key Words: Procaine, cellular aging, growth rate, long term treatment, enzyme thermolability

INTRODUCTION MANKIND HAS always tried to retard the aging process with plant extracts and more recently with synthesized chemicals. Procaine is one of these reported active compounds and was first developed as a local anesthetic. It was later introduced under the trade name Gerovital for use in elderly patients (Asian et al., 1965). U n d e r experimental conditions, procaine has been reported to have increased both the mean and maximum life span of male rats (Asian et al., 1965) and therefore has been considered as a possible antiaging molecule. Many authors have challenged this conclusion and have shown that the main therapeutic effect of procaine consists in reducing disease vulnerability rather than slowing down the aging process (Finch and Hayflick, 1977; Kent, 1982). The mechanism of action of this molecule is also a much debated subject. Local anesthetics act by blocking the Na + and K + transport in nerve cells and procaine behaves in a similar way (Ritchie and Greene, 1980). H o w e v e r , it is possible that the antiaging effect could result from another type of action (Kent, 1982; Thomas, 1983; Finch and Hayflick, 1977). Kent (1982), for example, proposed that due to an inhibitory effect on neuronal monoamine oxidase, the psychophysiological performance of the elderly improved under procaine treatment. C o n t r o v e r s y on the action of procaine led us to study its effect on a well-defined and reproducible model of cell aging, i.e., the in vitro aging of (Received 21 February 1986;Accepted 23 July 1987)

87

88

E. PIGEOLET et a/.

human fibroblasts. The relationship between this model and in vivo aging has been extensively studied and well established (Hayflick, 1965; Martin et al., 1970; Hayflick, 1974; Goldstein, 1974). Apart from this reproducibility, minor effects of potential antiaging drugs can be more easily observed and the incidence of pathological diseases that complicate the interpretation of data in the whole animal is avoided. In this model of in vitro aging, procaine showed a short-term stimulatory effect on cell growth but the data do not support an antiaging effect of the drug.

MATERIALS AND METHODS Culture WI-38 fibroblasts were purchased from the American Type Culture Collection, culture medium from Eurobio (Paris, France), fetal bovine serum from Sera-Lab. (Sussex, GB), and aureomycin from Lederle S.A. (Brussels, Belgium). Plastic culture multidishes were obtained from Sterilin (Feltham, G.B.). Chemicals Procaine was purchased from Pharmachemic (Anvers, Belgium). Chemicals were of analytical grade and purchased from E. Merck (AG Darmstadt, F.R.G.) except labeled thymidine which was obtained from New England Nuclear (Boston, MA). NTB2 photographic emulsion and DI9 developer were purchased from Kodak Eastman (Rochester, NY, USA) and Ilford Hypam fixer from llford Ltd (Essex, G.B.). Substrates for glucose-6-phosphate dehydrogenase assays came from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Cell culture As described by Hayflick (1965), WI-38 cells were serially cultivated in Eagle's minimum essential medium and 10% fetal bovine serum in 75 cm 2 plastic flasks. Up to passage 43, cells grew exponentially and were considered to be in phase II as defined by Hayflick (1965). At the end of the culture, or phase III, cell growth slowed and cells stopped dividing after approximately 57 population doublings. For the growth curves, cells were subcultivated at a 1:4 dilution in multidishes consisting of 25 wells of 4 cm" containing 2 ml of culture medium each. During subcultivation, the protein content of each well was measured several times according to Lowry (Lowry et al., 1951). Cell number was determined by using a Coulter cell counter (Dunstable England). Each result is the average of three determinations obtained on at least 40,000 cells. Autoradiography Cells from a confluent culture were trypsinized, diluted 15 times and seeded in 6 cm diameter Petri dishes containing coverslips. In the test, procaine was added at a final concentration of 5 x 10 4 M. After an incubation period of 3 days, cells were cultivated for 24 h in the presence of radioactive thymidine (2 Ci/mmole and 1 p~Ci/dish) and then fixed with a methanol/acetic acid solution (3/I). The coverslips were removed, dipped into a KCr (SO&, (0.05%) and gelatin (0.5%) solution and then into a photographic

89

PROCAINE AND IN VITRO CELLULAR AGING

150

120

cO

60

0

-4.0

~, -\prot

I

I

-3.5

-3.0

-2.5

-2.0

Log. concentration (M) FIG. 1. Dose-response curve of" procaine on the growth of human WI-38 fibroblasts in culture. Cells at passage n u m b e r 31 were subcultivated at a I/4 dilution in multidishes: after 1 day. the m e d i u m was removed and replaced by m e d i u m containing increasing concentrations of procaine (for 6 days). Results are e x p r e s s e d as percentage of the protein content (©) or cell n u m b e r (O) of the control culture. Each point is the result of triplicate assays.

emulsion solution (Kodak NTB2). They were finally stored in a dry atmosphere at 4°C and developed after 3 weeks. G6PD thermoinactivation assay

The thermoinactivation curves were performed as described by Holliday and Tarrant (1972) on a cell supernatant prepared according to; Houben et al. (1984). G 6 P D i n a c t i v a t i o n at 4 ° C

Confluent cultures were harvested and incubated at 4°C in a 50 mM Tris-HCI pH 7.4 buffer containing 0.15 M NaCl. At regular intervals, aliquots were taken, homogenized and G6PD assayed (Houben et al., 1984). Procaine assay

Procaine was assayed according to the method described by Brodie et al. (1948). RESULTS The effect of procaine on cell cultures was first tested by a dose-response curve on cell growth. Figure 1 shows the effect of increasing procaine concentrations on both the protein content and cell number of the culture after an incubation period of 6 days. The maximum effect was observed for a concentration of 10 -3 M with a stimulation of 40%. At higher concentrations the molecule rapidly becomes toxic for the cells. In order to avoid any possible toxic effect, we performed subsequent experiments at a much

90

E. PIGEOLET

et a/.

A

0.6

/./f"

0.4

0.2

1210

E

v

c-

h_

0.6

B

0.4

0.2

0.0

0

I

I

2

4

I

I

I

I

6 8 10 12 Days after subcultivation

I

I

14

16

FIG. 2. Effect of procaine on the growth of p h a s e II (A) and phase 11I (B) Wi-38 fibroblasts in culture. Cells passage n u m b e r 41 (A) and 54 (B) were subcultivated at a 1/4 dilution in a culture m e d i u m alone (O) or containing 5 x 10 ~ M procaine (O). Each point is the average of three a s s a y s . The experiment was performed in a multidish.

lower concentration of 5 x 10 4 M (see below). Procaine assays were also performed in similar experiments. T h e y showed that 53% of the initial procaine was still in the culture m e d i u m even after 6 days. Procaine was also tested on the kinetics of cell growth and these results are presented in Fig. 2. The stimulatory effect of procaine on phase II cell growth was very clear throughout

PROCAINE AND IN VITRO CELLULAR AGING

91

2.0

=

.

O

"E 1.0

0.5 55

i 70

a 85

Lifespan completed

i 100

(%)

FIG. 3. Evolution of the labeling index of cells expressed as the percentage of unlabelled nuclei in a continuous cell culture (0). At some passages, cells were first subcultivated at 1/15 dilution for three days with procaine 5 x 10 4M (O) before the incubation of 1 day with the [:~I] thymidine. Each point is the counting of at least 600 cells. The dashed line represents the results of Cristofalo and Sbarf (1973). the culture period [Fig. 2(A)] and reached its m a x i m u m on the 1 lth day. In the phase II1 cultures, however, the difference between the control and the diet test was negligible [Fig. 2(B)]. Because the effects of procaine on phase II and phase III cells were contradictory, we systematically studied its effect on cell division by following the mitotic index (Cristofalo and Sharf, 1973) in a continuous cell culture. Therefore, cells at population doubling 34 were serially cultivated until the termination of cell replication. At every 2 passages, 2 flasks were withdrawn and cultivated for 3 days with or without procaine at 5 x 10 -4 M and then for 24 h with radioactive thymidine before fixing and treating the cells for autoradiography. In the conditions of this assay, cultures did not reach confluency but cell density was slightly higher when procaine was present. The percentages of cells that did not incorporate labeled thymidine during the 24-h period are given in Fig. 3. Results clearly show a general reduction of D N A synthesis capacity due to culture age. Cells cultivated with procaine divided more actively than did control cells. This difference, h o w e v e r , diminished in the latest passages. The stimulatory effect of procaine is therefore dependent on age and is more p r o n o u n c e d in phase II than in phase Ill cells. The long term effect of procaine on cell culture is reported in Fig. 4. Cells were serially cultivated from population doublings 35 to 57, until they stopped dividing. When procaine was added to the medium, an e n h a n c e m e n t of cell proliferation was first o b s e r v e d but finally cells stopped dividing several population doublings before the control. T h e r e are at least two plausible explanations: (1) the continuous presence of

92

E. PIGEOLETel

al.

55

o/ .o:o

oj, J °

o

.(f/ ,/z 35n 0

n 40

0 80

Time in culture (days) FUG. 4. Long term effect of procaine on cell culture. Cells were subcultivated at a 1/4 density from passage 35 until the arrest of the cell divisions either, as described in materials and methods, in a normal medium alone (2]) or containing procaine at 5 × 10 ~ M (m), 2.5 × 10 M (C) and 1 × [ 0 ' M (O). The data give the passage number according to the time of subcultivation.

procaine is either toxic for the cells or (2) it accelerates cell metabolism, resulting in an early stimulation of turnover and replication, followed by " p r e m a t u r e " aging. This second idea, though it cannot be ruled out, does not fit in with experimental observations. First, growth stimulation was not exceptional and as soon as the cells became confluent they were subcultivated to avoid shedding as can be observed in Fig. 2A. Second, morphological observations (not shown) also confirmed the toxic effect of procaine. We observed an accumulation of lipid residues and lipofuscin, RER vesiculation and mitochondrial swelling. Finally, the data with procaine are in agreement with previous experiments performed with vincamine (Remacle, 1982): vincamine also stimulates cell growth at low concentrations, without modification of the total population doublings; however, at higher concentrations, cell growth initially stimulated, was followed by long term toxicity. In order to conclude this investigation, we wanted to test the possible influence of procaine on the appearance of a well-known biochemical marker for cellular aging, i.e., the accumulation of modified enzymes. Holliday and Tarrant (1972) and H o u b e n et al. (1984a, b) have shown that thermolabile G6PD accumulated in cultivated aging (phase III) human fibroblasts, whereas this thermolabile form was absent in young (phase II) cells. The appearance o f aberrant, thermolabile, enzyme molecules may therefore be considered as an index of cellular aging. Figure 5(A) shows the termoinactivation curve o f G6PD obtained on a supernatant of phase II cells, fitting with a straight

93

PROCAINE AND IN VITRO CELLULAR AGING

100~ A

5O

o_

20 ¸

100i

50"

r,3

20-

10

I

I

10 20 Incubation time at 60 ° C (min)

.I

30

FIG. 5. Thermoinactivation of G6PD in phase II cells (A) and effect o f procaine on the a m o u n t o f thermolabile G6PD in p h a s e llI cells (B). Cells from passage 36 were cultivated for 7 days without procaine (A). Cells from passage n u m b e r 56 were incubated at a 1/2 dilution for 9 days with (O) or without ( 0 ) procaine at a 5 x 10 4 M concentration. The m e d i u m was c h a n g e d after 7 days (B). Cells were then harvested, homogenized and supernatants heated at 60°C. T h e residual G6PD activity was a s s a y e d and is expressed as the percentage o f the initial values on a logarithmic scale.

94

E. P I G E O L E T et ~d.

line, which supports the existence o f o n e enzymatic population. In Fig. 5(B), the same experiments have been performed on phase III cells incubated with or without procaine. The nonlinearity of the curves suggests an heterogeneity in the enzymatic population, with the appearance of a more thermolabile fraction. This enzymatic fraction is similar in both cases and accounts for 10% of the total e n z y m e activity. We can thus conclude that procaine has no effect on the amount of thermolabile G6PD present in phase III cells. DISCUSSION Results of the effect of procaine on phase |1 cells in culture seemed initially promising since they indicated a clear increase in growth rate and in protein content (Figs. 1 and 2). The stimulation of cell division was also obtained in serially cultivated cells in the presence of procaine but they all stopped dividing many population doublings before the control in a concentration dependent manner. We can therefore conclude that procaine has a long term toxic effect, even at concentrations much lower than expected from the dose-response experiments on cell growth (Fig. 1). In our work procaine did not lead to an increase in population doublings in cell cultures and to date very few substances have been shown to have such an effect (Macieira-Coehlo, 1966; Cristofalo, 1970; Todaro and Green, 1964). One well-known biochemical m a r k e r for in vitro cell aging is the presence of thermolabile G6PD. The mechanism leading to the appearance of a more thermolabile fraction, has been extensively studied by Houben et al. (1984a and b) and Fundu e t a / . (1985). These authors have shown that the labile enzyme is an intermediary form between the active dimer and the inactive monomer. Many factors can affect this equilibrium and this is probably why the presence of labile G6PD has been questioned in the literature (Pendergrass et a l . , 1976). In the present experiments phase III cells were found to contain 10t~ of thermolabile G6PD. Procaine did not affect this percentage. Vincamine, on the other hand, could stimulate the growth of phase 111 cells and reduce the amount of thermolabile G6PD and superoxide dismutase (Houben et a l . , 1984b; Somville et a l . , 1985). This, however, does not occur with procaine. From the data presented in this paper we could not see any positive effect due to procaine on phase III cells. The mechanism of action of procaine is still a matter of controversy. As a local anesthetic, it can act on the nervous system like any other anesthetic by decreasing sodium transport and thus the membrane polarization (Lee, 1976). It therefore interferes with the functions of organs in which conduction or transmission impulses occur. This could, eg., explain procaine's action on the cardiovascular system (Ritchie and Greene, 1980). Observations on the effect of procaine on cells show that it modifies a series of processes which are difficult to explain only by interference with sodium transport. For example, Freim/Jller et al. (1980) have shown that there is an increase of glutamate dehydrogenase and glutamate oxaloacetate transaminase activity on isolated heart and skeletal muscle cells. They also found that the presence of procaine inhibited thymidine incorporation in these cells whereas a pretreatment had a stimulatory effect. Other reports have shown that it could not only protect mitochondria in vitro (Johnson et al.. 1972) but also mitochondria in old rats (Bai et al., 1984). At low concentrations procaine could also stabilize microtubules (Goffe et al., 1985) and regulate genetic expression

PROCAINEAND IN VITROCELLULARAGING

95

(Granett and Villarejo, 1982). Although the exact mechanism of action of procaine is not well defined, we report here a significant effect of this molecule at low concentrations on the replication of phase II cells. However, this positive effect gradually disappeared as the cultures reached phase III (Fig. 3) and completely vanished in the latest passages. We were not able to find any positive effect on phase III cells. In the model of in vitro aging used, we propose procaine to be a transient metabolic stimulator rather than an antiaging molecule.

REFERENCES ASLAN, A. Novocaine als eutrophischer Faktor und die M6glichkeit einer VerKingerung der Lebensdauer. Ther. Umschau. Bern. 9, 165-173, 1956. ASLAN, A., VRABIESCU, D. CAMPEANU, C., and STANESCU, S. Long term treatment with procaine in albino rats. J. Geront 20, I-8, 1965. BAI, F.F., MIGHEL, R., and ROSSIGNOL, P. Effects of procaine on the oxidative phosphorylation of brain mitochondria of senescent rats. Mech. Age. Dev. 26, 277-282, 1984. BRODIE, B.B., L1EF, P.A. and POET, R. The fate of procaine in man following its intravenous administration and method t\)r estimation of procaine and diethylaminoethanol. J. Pharmacol. [~5.vp. Ther 94, 359-366, 1948. CRISTOFALO. V.J. In: Aging in Cell and Tissue Culture. Holeckova, E. and Cristofalo, V.J. (Editors), pp. 83-119. Plenum Press, London. 1970. CRISTOFALO, V.J. and SHARF, B.B. Cellular senescence and DNA synthesis: thymidine incorporation as a measure of population age in human diploid cells. Exp. Cell Res. 76, 419-427. 1973. FINCH, C.E. and HAYFLICK, L. Life table modification and life prolongation. In: Handbook ~/'the Biology ~f'Aging, pp. 626-627, Van Nostrand Reinhold, New York, 1977. FREIMULER, VON B., MULLER. W.H., and LAMPRECHT, W. Einflul~ yon Procain und Nicotinoyl-procain auf Enzymaktivitaten, N ucleins~iure und Proteinsynthese bei Prim~ikulturen yon Herz- und Skelettmuskeizellen der Ratte. Arzneim.-Forsch. 30, 1641-1643. 1980. FUNDU, D.L.M., HOUBION, A., and REMACLE, J. Alteration of enzymes in aging human fibroblasts in culture. IV. Effect of glutathione on the alteration of glucose-6-phosphate dehydrogenase. Mech. Age. Dev. 32, 249-266, 1985. GOFFE, G., FOUCAULT, G. RAYMOND. H.N., and PUDLES, J. Dual effect of procaine in sea urchin eggs. Exp. Cell Res. 15, 175-181, 1985. GOLDSTEIN, S. Aging in vitro: growth of cultured cells from the galapagos tortoise. Exp. Cell Res. 83, 297-302, 1974. GRANETT, S. and V1LLAREJO. M. Regulation of gene expression in Escherichia coli by the local anesthetic procaine. J. Mol. Biol. 160: 363-367, 1982. HAYFLICK, L. The limited in vitro lifetime of human diploid cell strains. Exp. Cell Res. 37,614-636, 1965. HAYFLICK. L. The longevity of cultured human cells. J. Am. Gerontol. Soc. 22, 1-12. 1974. HOLLIDAY, R. and TARRANT, G.M. Altered enzymes in aging human fibroblasts. Nature 238, 26-30. 1972. HOUBEN, A,, RAES, M., HOUBION, A., and REMACLE, J. Alterations of enzymes in aging human fibroblasts in culture. 1. Conditions for the appearance of an alteration in glucose-6-phosphate dehydrogenase. Mech. Age. Dev. 25, 25-35, 1984a. HOUBEN, A., RAES, M., HOUBION, A., and REMACLE, J. Alterations of enzymes in aging human fibroblasts in culture. II. Conditions for the reversibility and the mechanism of the alteration of glucose-6phosphate dehydrogenase. Mech. Age. Dev. 25, 35-45, 1984b. JOHNSON, C.L., GOLDSTEIN, M.A., and SCHWARTZ, A. On the molecular action of local anesthetics. I. The mitochondrion as a model membrane system for studying local anesthetic action. Mol. Pharmacol. 9, 360-371, 1972. KENT, S. The procaine "Youth" drug. Geriatrics 37, 32-36, 1982. LEE, A.G. Model for action of local anesthetics. Nature 262, 545-549, 1976. LOWRY, O.H., ROSEBROUGH, N.J., FARR, A.L., and RANDALL, R.J. Protein measurement with folin phenol reagent. J. Biol. Chem. 193, 265-275, 1951.

96

E. P[GIEOLETet al.

M A C I E I R A - C O E H L O , A, Action of cortisone on h u m a n fibroblasts in vitro. Experientia 22, 390-391, [966. M A R T I N , G.M., C U R T I S . A,, S P R A G U E , B.S., and E P S T E I N , C.J. Replicative life-span of cultivated h u m a n ceils. Lab. Invest. 23, 86-92, 1970. M U R D O C H R I T C H I E , J. and G R E E N E N I C O L A S , M. Local Anesthetics. In: Goodman Gilm(m's the Pharmacological Basis o f Therapeuti( s, 6th Ed., pp. 300-308, MacMillan Publishing, New York, 1980. N A G Y , 1.Zs. and SEMSEI, 1. C e n t r o p h e n o x i n e increases the rates of total and m R N A synthesis in the brain cortex of old rats: an explanation of its action in terms of the m e m b r a n e hypothesis of aging. Exp. Gerontol 19, 171-178, 1984. P E N D E R G R A S S , W.R., M A R T I N , M.G., and B O R N S T E I N , P. Evidence contrary to the protein error h y p o t h e s i s in vitro s e n e s c e n c e . . I . ('ell Physiol. 87, 3-13, 1976, R E M A C L E , J. Evolution, plasticitd et modifications des cellules au c o u , s du vieillissement. Effet de la vincamine sur le vieillissement cellulaire. Ed. Ag(,n('e M.K. Pari.~ 63, 1962. S O M V I L L E , M., H O U B E N , A., RAES, M., H O U B I O N , A., H E N I N , V.. and R E M A C L E , J. Alterations of e n z y m e s in aging h u m a n fibroblasts in culture. Ill. Modification of superoxide d i s m u t a s e as an environmental and reversible process. Mech. A~,('. 1)ev. 29, 35-5[, 1985. T H O M A S , R. Procaine. will it keep you y o u n g e r Io1~ger? M('d..I. Attsl. i 1,543-545, t983. T O D A R O , G.J. and G R E E N , M. Serum albumin supplemented medium for long term cultivation of m a m m a lian fibroblasts strains. Proc. Soc. Evp. Biol. Med. 116, 688-692, 1964.