Studies on the effect of tetrandrine on microtubules

ECOTOXICOLOGY

AND

ENVIRONMENTAL

SAFETY

15,142-148

( i 988)

Studies on the Effect of Tetrandrine

on Microtubules

I. Biochemical Observation and Electron Microscopy LINGF-EN LIU, NINGMENG CHEN, GUOPING JIGONG YANG, AND YURUI

CAI, ZHAOLIN LI

LI,

Department ofPneumoconiosis, Institute of Occupational Medicine, Chinese Academy ofpreventive Medicine, 29 Nan Wei Road, Beijing, China Received March 17,1987 Microtubules were purified by using two cycles ofassembly and disassembly processes on fresh brain homogenates from 30 guinea pigs. The yield was about 60 mg. The effect of tetrandrine on tubulin was determined by spectrophotometric analysis and electron microscopy. In addition, we used the indirect immunofluorescent method including tubulin antibody to locate the presence of microtubules in 3T3 cells by fluorescence microscopy. The effectsof colchicine and Pzo4 were studied for comparison at the same time. The results showed that colchicine can effectively depolymerize microtubules, while tetrandrine showed aggregation, and in a different manner. The shape and structure of microtubules were definitely destroyed by colchicine, but were not affected by Pzo4which protected against the destructive effect of tetrandrine. This result indicated the safety of using a combination of P204and tetrandrine in the treatment of silicosis. o 1988 Academic

Press, Inc.

Tetrandrine (TT) exhibits many pharmacological reactions. It has been used effectively in the treatment of high blood pressure and cardiopathy, among others. Recently, we found that TT showed an inhibitive effect on experimental silicosis of rats (Yu et al., 1983). Chest X-rays of some silicotic patients treated with TT for l-3 years showed obvious changes; the silicotic nodules became smaller and shadows became clearer (Lu et al., 1983). It is known that colchicine inhibits microtubule polymerization in vitro and in vivo, so that elimination of procollagen from the cells by the movement of the cytoskeleton was hindered and the biosynthesis of collagen was blocked at this step (Liu et al., 1985). In this report we attempt to show whether TT has the same capability as colchitine to interupt the process of collagen biosynthesis. Tubulin was first purified from guinea pig brain and reacted with TT in vitro. The effect of TT on tubulin polymerization was observed by spectrophotometric analysis and electron microscopy, and the effect of colchicine was studied at the same time. MATERIALS

AND

REAGENTS

(1) Guinea pig brains. (2) Buffer solutions. (a) Stabilizing buffer solution-O. 1 A4 Mes, 1 mM EGTA, 0.5 mM MgC12, 4 Mglycerol, pH 6.6; (b) polymerizing buffer solution-O. 1 M Mes, 1 mMEGTA, 1 mMGTP, 0.5 mMMgC&, 8 Mglycerol, pH 6.4; (c) depolymerizing buffer solution-O. 1 mMMes, 1 WEGTA, 0.5 mA4MgC12, pH 6.6.

0147-6513188 $3.00 Copyright 62 1988 by Academic Press, Inc. AU rights of reproduction in any form reserved.

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METHODS 1. Purijication of microtubules. The purification of microtubules was based on the method of Shelanski et al. (1973) by use of two cycles of assembly and disassembly processes on fresh brain homogenates from 30 guinea pigs (body wt about 500 g). The brain tissues were minced and washed, and the suspension was decanted through eight layers of cloth. Of the precipitates, 85 g (wet wt) was homogenized in stabilizing buffer solution (1 ml/g wet wt) with a Waring blender at the maximum setting for 30 set at 4°C and with two strokes of a motor-driven glass-Teflon homogenizer operated at maximum setting. The homogenate was centrifuged in a Beckman L8 ultracentrifuge at 100,OOOg for 60 mitt, at 4°C. The supernatant was mixed with an equal volume of a mixture containing the buffer, incubated for 30 min in a 34°C water bath, and then centrifuged at 100,OOOg for 60 min, at 30°C. The volume of the warm supemate was recorded and the supernate was discarded. The microtubule pellets were resuspended in an ice-cold depolymerized buffer to make a volume equal to 25% of the volume of the warm supemate. The pellets were then removed in pieces, from the bottom of each tube by gentle scraping with a rub ber-tipped stirring rod. The resulting coarse suspension was poured into an ice-cold, motor-driven glass-Teflon homogenizer and subjected to about five gentle strokes to produce a uniform suspension. This suspension was placed at 0°C for 30 min to depolymerize the microtubules. The suspension was centrifuged at 100,OOOg for 60 min, at 4°C. The supernatants were carefully removed from the pellets. A second cycle of polymerization and centrifugation was carried out by incubating the solution at 34°C as described above. The yield was about 60-mg microtubule pellets which were stored at -80°C after being rapidly frozen in liquid nitrogen. The protein content was determined by the Lowry method (Lowry et al., 195 l), and bovine serum albumin was used as standard. A scheme of the procedure for purification of microtubules is expressed in Fig. 1. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein fractions were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) using a slight modification of the method of Palmiter et al. ( 197 1). Samples (60 rg) were added to 5 mg SDS, 40 mg sucrose, and 10 ~1 mercaptoethanol, heated for 30 set in a 100°C water bath, and run on I 1% gels. 3. Spectrophotometric analysis. The polymerization reaction was followed by measuring the increase in turbidity which results from microtubule formation and was monitored at 350 nm in a Beckman DV Model 8 spectrophotometer equipped with recorder (see Zaremba et al., 1984). Protein samples (1 ml) were placed in cuvettes on ice, GTP (guanosine S-triphosphate, final concentration 1 r&d) was added and mixed, and the increase in turbidity at 350 nm was recorded as a function of time at 37°C. TT (0.066-o. 1 mM) or colchicine (0.1 mM) was added separately to the tubulin samples before and after polymerization. 4. Electron microscopy. Drops of the microtubule solution (samples treated as mentioned above) were placed on the grid coated with 0.25% Formvar, allowed to stand for 3 min, rinsed with stabilizing buffer solution, and blotted. The grids were stained with 2% phosphotungstic acid, 1.5% uranyl acetate was placed on the grid and allowed to stand for 15 min, and the grids were again blotted and allowed to dry.

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brain

Homogenized Centrifuge

at

tissues

c

at 4’~ t

100,000

for

g,

10

4OC

8ec

for

60 mln

1

9 pellet

1

Added equal volume of polymerized buffer solution, placed on the warm water bath (34’C for 30 min, g. 30°C for 60 min. centrifuge at 100,000 pellet Ground

buffer

pellets with depolymerieation eolution (the volume of it was

the

25 % of eupemate placed at O°C for

100,000

-product

1

(2). the euepeneion 30 min. centrifuge

uau at

g, 4’C for 60 min.

FIG. 1. Scheme for the purification of microtubule proteins by cycle of temperature-dependent ization anddepolymerization.

RESULTS

AND

polymer-

DISCUSSION

1. SDS-PAGE. The tubulin was stained with Coomassie brilliant blue (Fig. 2), and the purity was identified by SDS-polyacrylamide gel electrophoresis. More than 85% of the protein coincides with tubulin. The upper bands were high molecular weight associated protein (MAPS). The lower bands were two monomers ((wand ,f3tubulins). These two tubulins have molecular weights of 60,000. A small amount of minor components was usually present (Fig. 2). 2. Spectrophotometric analysis. Figure 3a shows the patterns of process of polymerization of 1 ml tubulin (2 mg/ml), tubulin treated with TT (0.066-o. 1 mM), and tubulin treated with colchicine (0.1 mM) from 0 to 37°C for 30 min. The drugs were added before polymerization occurred. Results of tubulin and tubulin treated with TT were similar (Fig. 3a, 1 and 2). The optical absorption or turbidity of the solutions

> MAPS

- TUBULIN

FIG. 2. SDS-polyacrylamide gel (11%) of microtubule protein after two cycles of in vitro purification by assembly-disassembly. MAPS and tubulin are indicated.

EFFECTS OF TETRANDRINE

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(min)

FIG. 3. Effect of TT and colchicine on polymerization of tubulin. 1, Tubulin; 2, tubulin + TT; 3, tubulin + colchicine. (a) Drugs added before polymerization. (b) Drugs added after polymerization. (4) Start to add drugs.

was gradually increased while the process of polymerization was going on and reached maximum after 30 min. By adding colchicine to tubulin, the process of polymerization was hindered (Fig. 3a, 3) and the optical absorption did not increase as much as that for the untreated tubulin or tubulin treated with TT. The effects of TT and colchicine on polymerization were also studied by adding the drugs to tubulin after it was polymerized (Fig. 3b). When TT was added, the pattern was similar to that in Fig. 3a; a very small decrease in optical absorption was seen in Fig. 3b, 1 and 2; but with colchicine, the optical absorption decreased immediately, indicating a marked depolymerization (Fig. 3b, 3). When TT was added up to 0.02 mM (or on standing over night) aggregation could be observed which showed that TT can combine with microtubules. 3. Electron microscopy. Microtubules obtained by multiple cycles of assembly and disassembly in vitro contained both tubulins and MAPS. When examined under the electron microscope, microtubules were present in long thread-like forms crossing each other, the entire surface being decorated with filamentous or fuzzy material

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FIG.

4. Tubulins were assembled to microtubules at 37°C for 30 min. X29,000.

FIG.

5. Microtubule

FIG.

proteins in cold environment (0°C). X75,000.

6. Tubulins + TT at 37’C.

X57,ilo.

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FIG. 7. Tubulins + TT (concn above 0.2 m&f) at 37°C over night. X43.000.

(Weisenberg, 1972) (Fig. 4). Microtubules were disassembled and turned to ringshaped structures (Fig. 5) in a cold environment (OOC) or when colchicine was added. By adding TT (0.066-o. 1 mM) to microtubules, the filamentous materials remained but became more aggregated (Fig. 6). When TT was increased to 0.2 m&I and the mixture of aggregates was examined under the electron microscope (Fig. 7) it lost the filamintous form, and the ring-shaped, depolymerized structure was also observed as with colchicine (Fig. 8) or under 0°C. CONCLUSION From the above results, it was suggested that colchicine can effectively depolymerize microtubules, while TT showed slight depolymerization activity. The reaction is slow and occurs somewhat differently. TT coprecipitated with microtubules which was similar to the reaction of vinblastine forming paracrystals with microtubules (Clarkson et al., 1986).

FIG. 8. Microtubules were depolymerized by colchicine. X29,000.

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REFERENCES T. W., SYVERSEN, T. L. M., AND SAGER, P. R. @Is.)(1986). TheCytoskekton: A Targetfor ToxicAgents,pp. 26-27. Plenum, New York. LIU, L. F., etal. (1985). Inst.Health14(4),1. LOWRY, 0. H., etal. (1951).J. Biol. Chem.193,265. Lu, X. R., etal. (1983). Chin.J. Znd.Hyg.Occup.Med.12(2), 1. PALMITER, R. D., etal. (197 1). J. Biol.Chem.246,742. SHELANSKI, M. L., etal. (1973). Proc.Natl.Acad.Sci. USA70(3),365. WEISENBERG, R. C. (1972). Science 177,1104. Yu, X. F., etal. (1983).Ecotoxicol.Environ.Saf:7,306. ZAREMBA, T. G., et al. (1984). Biochemistry23(6), 1073. CLARKSON,