Effect of 15-azasteroid analogues on cell culture growth

301

EFFECT OF 15-AZASTEROID ANALOGUES ON CELL CULTURE GROWTH M. Louise Higgins', Robert W. Chesnut2, Franklin R. LeachI, John G. Morgan3, K. Darrell Berlin3, and Norman N. Durham' Departments of Biochemistr?, Microbiolog?, and Chemistrys, Oklahoma State University Stillwater, Oklahoma 74074 Received:12/6/71

ABSTRACT Two azasteroid analogues, l,lO,ll,lla-tetrahydro-lla-methyl-2Enaphth[l,2-L] indol-y-01 (compoundI), and l,lO,ll,lla-tetrahydro-~methoxy-lla-methyl-2g-naphth[l,2-E] indole (compound II) inhibit the growth of KB and L-M cells in culture. There is a structural specificity for biological effectiveness with compound I being more inhibitory at equivalent concentrations. When compound I is present during growth of KB and L-M cells, the permeability of the lysosomal membrane is increased. Compound I reduced in a nearly equivalent manner the transport of precursors into the cells for all three macromolecular species, protein, DNA and RNA. The action of compound I can be accounted for by an action upon the cell membrane. INTRODUCTION Steroid hormones regulate many diverse metabolic reactions and processes such as glycolysis and gluconeogenesis, the immune response, electrolyte homeostatis and blood pressure, spermatogenesis, ovarian processes, lipid metabolism, and fatty acid synthesis and catabolism (see review 1). Many estrogenic steroids inhibit cell growth _in vitro as demonstrated by work with chick-heart fibroblasts, dental pulp, bone marrow, and rabbit spleen cells (2).

Synthetic estrogens (3,4)

in-

hibit a microbial glucose-6-phosphate dehydrogenase competitively with respect to glucose-6-phosphate and noncompetitively with respect to nicotinamide adenine dinucleotide phosphate.

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Equilenin has been isolated from the urine of pregnant mares.

HO It has female hormone activity (5),

and has also been isolated from

human adrenal carcinoma (6). Several nitrogen-containing synthesized in our laboratory (7).

analogues of equilenin have been Insertion of nitrogen greatly

increases the solubility of the steroid which may be important for biological activity.

We wish to report the results obtained with two

of these compounds, 1,10,11,lla-tetrahydro-lla-methyl-2~-naphth[1,2-~] indol-T-01 (compound I) and l,lO,ll,lla-tetrahydro-7-methoxy-llamethyl-2E-naphth[l,P-g]indole

Compound I

(compound II) for the biological

Compound II

activity with respect to growth, stimulation or inhibition, permeability of cells to precursors and substrate for lysosomal enzymes, and

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'303

biosynthetic capabilities of KB and L-M cells cultured -in vitro. Chesnut and co-workers (8) reported both compound I and II inhibited microbial growth and showed a synergistic antimicrobial action with chloramphenicol, actinomycin D, polymyxin and circulin.

Cell cultures

The KB cells, originally obtained from Dr. Vernon Scott, University of Oklahoma Medical School, were grown as monolayer cultures at 37°C in either milk dilution bottles or Falcon plastic tissue culture dishes, using medium 199 supplemented with 10% calf serum. L-M cells, purchased from the American Type Culture Collection Cell Repository, were cultured in 125 ml Erlenmeyer flasks containing 20-30 ml of McCoy's 5a modified medium supplemented with 6$ calf serum on a New Brunswick G-10 gyrotory shaker at 50 os~i~~ations/min in a 57°C controlled temperature room. The media were purchased from Grand Island Biological Company and the serum from Microbiological Associates. Cell growth and plating efficiency The cells were trypsinized, and susp:nded in medium. Cell numbers were determined by using a Coulter cell counter. The KB ce1l.s(500-600) were plated in 60 x 15 mm Falcon plastic tissue culture dishes containing a total of 5 ml of medium, Cells ore grown in a CO, gas phase incubator at 37°C for 7 days. The medium was removed, the plates washed with Hanks' salt solution, and the cells stained with a 0.5s aqueous crystal violet solution, rinsed and dried. The colonies were counted microscopically and the relative plating efficiency calculated using the control value of lOO$, Cells prepared in this manner were also studied microscopically for morphological changes. Flasks were inoculated with 1 x lo5 L-M cells/ml and growth determined by removing 0.2 ml aliquots at varying times and counting the cells, For demonstrating the reversibility of the inhibition, L-M cells were exposed to 20 ug,/mlof compound I until inhibition was apparent as compared to the control (36 hr). The medium was removed, cells washed with medium, and suspended in medium without: the compound. The number of cells in the control were reduced to 1.2 x lo5 cells/ml, which was equivalent to the titer of the inhibited culture.

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The acid phosphatase in lysosomes of unfixed FB and L-M cells on coverslips was determined by histochemical staining (9). The cells were grown on coverslips in medium 199 with 10% calf serum. For the 24 hr growth prior to staining, no compound, or compound I or compound II (20 &g/ml) was added. The coverslips were washed in Hanks' salt solution, incubated at 37°C in Gomori's substrate solution for 1, 2, or 3 hr washed, treated with 1% solution of ammonium sulfide, washed, mounted, and observed microscopically. Light staining showed only a few scattered granules; medium staining was more dense but distinct isolated granules were still detected; and heavy staining showed more intense staining with fewer discrete granules. Adenosine 3',5'-cyclic phosphate (P-L Biochemicals) was added at a concentration of 1 x 10m6 M to L-M cells in suspension in McCoy's 5a modified medium with 6% calf serum which contained no addition, or compound I or compound II (10 ~g/ml). 3H-Thymidine, 3H-uridine, or 14C-leucine (Schwarz Bioresearch) were used to measure uptake and macromolecular syntheses. L-M cells (8 x LO5 cells/ml) were incubated in 20 ml of McCoy's 5a modified medium with shaking at 100 oscillations/min in a 37°C water bath in the presence or absence of compound I (15 yg/ml). 3H-Thymidine (10 PC), 3H-uridine (10 ~c), or 14C-leucine (5 MC) were added to a flask, one ml samples removed at varying intervals and the rates of uptake, and incorporation determined fromthe linear portion of the radioactivity versus time curves. Samples were put into tubes containing 3 ml of ice-cold phosphate buffered saline (PBS), centrifuged, washed with cold PBS and dissolved in 1 ml of Hyamine (Packard Instrument Company). Determination of the radioactivity retained in the cells yields the "uptake". An equal volume of cold 10% trichloracetic acid (TCA) was added to a duplicate sample, which was centrifuged, washed with 51 TCA, and dissolved in 1 ml of Hyamine to determine the inThe difference between the corporation into macromolecules. "uptake" and the "incorporation" is the "pool". Radioactivity was determined in a liquid scintillation spectrometer using Bray's scintillation fluid (10). RESULTS

Growth inhibition Growth,of both the KB and L-M cell lines was inhibited when the cells were grown in media containing 10 pg/ml. or greater concentrations of either compound I or II (Fig. 1).

Cultures containing

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305

.

6 4

Compound I O-O none or 1 @g/ml 4-O 10 kg/ml 6-e 20 bg/ml

// 0

o

/

k I

I

I

I

I

Y

O-O

I

none or

2

I

i

0

I

I

2

I

4

I

I

6

DAYS Fig, I.

Effect of varying concentrations of compound I and Compound II on L-p/Icell growth,

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1 pg/ml of either compound I or II had the same growth rate as the control.

During the first two days of growth, 10 fig/ml of either

compound reduced the growth by 25 to 50 percent.

By the 4th day

cultures containing 10 yg/ml of compound I remained at 50% of the control while cultures containing compound II showed only a 30% reduction in growth as compared to the control.

Increasing the con-

centration of compound II to 20 ug/ml depressed the growth rate only slightly over the rate observed with 10 pg/ml.

However, when

the concentration of compound I was increased to 20 pg/ml, a marked inhibition was observed with only a slight increase in cell numbers after day three. To ascertain if the potency of compound II were reduced during the incubation in the growth medium in the absence of cells, compound II (20 pg/ml) was added to the medium and incubated at 37°C on the shaker for 4 days.

Cells (1 x lO'/ml) were added to

the "incubated" compound-containing medium.

The inhibition pattern

obtained from the "incubated" medium was identical to that obtained when compound II (20 pg/ml) was added simultaneously with the inoculation.

These results show that detoxification of the compound

by either auto-oxidation or enzymatic processes associated with the serum does not occur during continued incubation in the culture medium. When L-M cells were exposed to compound I (20 pg/ml) for 36 hr and then the compound removed, the cells resumed normal growth within 48 hr thus demonstrating the reversibility of the inhibition (Fig. 2).

I

I I I :ompound I ,0-O none 8-6 20 fig/ml 8-e compound

0 Pig. 2.

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I

I

6

2 iDAYs4

Reversal of growth inhibition by removal of compound I after 36 hours.

The greater sensitivity of cells to compound I was also evident with KB monolayer cultures (Table 1).

The colony formation of KB

cells (7 days cultures) showed a 38% relative plating efficiency in the presence of 10 pg/ml of compound I and a 17% relative plating efficiency when 20 pg/ml of compound I was added to the growth medium. In contrast, compound II produced only a very slight inhibition of growth when compared with the control culture, with no significant difference observed between the 10 and 20 fig/ml concentration,

As

with L-M cells, 1 @g/ml oE either compound I or compound II had little effect on relative plating efficiency.

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TABLE 1 Effect of Compound I and II on KB Cell Growth

Relative plating efficiency ($) Concentration fig/ml Treatment

0

1

10

20

None

100

Compound I

___

91

38

17

Compound II

-__

97

82

79

Colony and cell morphology KB cells exposed to 10 and 20 @g/ml of compound I did not divide or, if limited division occurred, small colonies were formed containing only a few cells. Differences in cell morphology were also observed. In contrast to the control, the nuclei of treated cells were located at the periphery of the cell with little cytoplasm between the eel2 membrane and nucleus. In addition, the cytoplasm of treated cells had a less uniform density with areas of heavy and light staining. Since the difference in staining density could be a modification of intracellular structures such as lysosomes, experiments were performed to determine the effect of the compounds on the permeability of lysosomal membranes. Effect on permeability of lysosomal membranes The acid phosphatase was measured in BB and L-M cells grown in medium in the presence or absence of compound I or II (20 ,tig/ml)

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for 2,or 24 hr prior to staining (Table 2).

Quantitative evaluation

TABLE 2 Relative Acid Phosphatase Staining of KB and L-M Cells Grown in the Presence and Absence of Compound I and Compound II Incubation time in substrate (Hours)

Treatment none

compound I

1

none

light

none

2

light

medium

light

3

medium

heavy

medium

compound II

of the relative staining intensity of the lysosomes was compared in the treated and untreated cells.

The staining intensity of the cells

treated for 2 hr was indistinguishable from untreated cells. differences were noted for cells grown for

24 hours.

However,

Cells grown

in the presence of compound I showed light staining after 1 hr incubation in the substrate while the control and compound II treated cells did not stain.

For all

3 substrate incubation times, the con-

trol and compound II treated cells showed identical staining reactions while compound I treated cells showed a more intense staining reaction. Thus, cells grown in the presence of compound I accumulate the substrate within the lysosomes much more rapidly than the control of compound II-treated cells.

Since the availability of the substrate

is the rate-limiting step in the phosphatase determination, compound I produces a modification.of

the lysosomal membrane which increases

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the permeability of the lysosome to the substrate.

The results also

emphasize the difference in biological activity of compound I and II type molecules which chemically are similar except at C-3.

Differences

in the biological activity have been observed when the compounds were tested using various bacteria (8).

In experiments not shown, the

treatment of cells with Triton X-100 resulted in the same final staining intensity as growth in the presence of compound I.

Thus, com-

pound I does not induce acid phosphatase. Addition of cyclic AMP Since CAMP has a defined role as a second messenger in the action of many hormones and is active -in vitro (11,12) studies were made to determine if CAMP effected the biological activity of the azasteroids or produced a similar inhibition.

L-M cells were incu-

bated in fresh medium in the presence and absence of compound I or II (10, 15 yg/ml) and/or (1 x 10m6 M) for 6 days.

The presence of

exogenous CAMP did not influence growth inhibition by the compounds and was not inhibitory alone. Effect on uptake and incorporation The uptake of radioactive thymidine, uridine, and leucine, precursors for DNA, RNA, and protein, respectively, was inhibited when suspension cultures of L-M cells were incubated in the presence of 15 fig/ml of compound I (Table 3).

The radioactivity present in the

pool for each of the precursors was reduced by about 66% showing that the inhibition was not specific and the decreased pool level

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could account for the decreased incorporation into the macromolecules that was observed. TABLE

3

Effect of Compound I (15 pg/ml) on Uptake and Incorporation of 3H-Thymidine, 3H-uridine and 14C-leucine by L-M Cells Rate Cpm/8 x lo5 cells/hr Substrate

Addition

3H-thymidine

none

4,200

compound I

2,000

1,300

none

8,400

4,000

4,400

compound I

3,000

1,400

1,600

none

9,600

6,000

3,600

compound I

4,000

2,800

1,200

3H-uridine

14C-leucine

Uptake

Incorporation

3,000

Pool

1,200 7oo

DISCUSSION The azasteroid analogue, compound I used at a concentration of

10

a/ml,

inhibited the -in vitro growth of RB and L-M cells.

When

the hydroxyl group at the C-3 position is methylated, the inhibitory activity decreases as is seen using compound II.

Although the con-

centrations used in these studies were readily soluble in the media, the water solubility of compound II is less than compound I at neutral pH and the melting point is also lower.

Whether the differ-

ences in physical properties of the two compounds is the basis for the observed differences in biological activity is not known, but

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312

there is a structural specificity observed for inhibition with compound I which is a closer analogue of equilenin.

A similar specificity has

been observed in the microbial systems (8). Because of the known effects of steroid hormones on transport processes and hence upon membranes

(13), the effects of the steroid

analogues on lysosomal enzyme activity as a measure of the lysosomal membrane integrity and upon substrate transport were determined. Compound I accelerated the histochemical staining for acid phosphotase presumably by increasing the permeability of the lysosomal membrane to the phosphatase substrate (Table 2).

As expected, compound II in

equivalent concentrations was inactive.

Many other compounds such

as detergents, aromatic alcohols, transquilizers, toxic proteins, and steroid hormones, and physical factors such as oxygen and radiation labilize lysosomes (14,15) by modulation of the lysosomal membrane. Since the effect of compound I appears to require a period of growth, the changes in the lysosomal membrane are not due to a detergent-like action which is observable a short time after addition of the detergent, Triton X-100. Table 3 shows that the amount of radioactive precursor taken into the cell pool was reduced equally by the presence of compound I regardless of the precursor used.

Since the incorporation of radio-

activity into macromolecular species was reduced similarly to the pool level, there is no specific effect on macromolecular synthesis. The influence is apparently on the transport of the precursors across the cell membrane which could account for the growth inhibition

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1972

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observed in these studies.

An alternate explanation is that leakage

of pool material because of increased permeability can be responsible for the decreased pool levels.

ACKNOWLEDGEMENTS Supported in part by: (FRL) United States Public Health Service Research Career Development Award 5KO3-CAO6487, Oklahoma Agricultural Experiment Station Project 1096, American Cancer Society, Grant IN-9lC. Journal Article 2396 of the Oklahoma Agricultural Experiment Station.

REFERENCES 1.

Mckerns, K. W., STEROID HORMONES AND METABOLISM. Century-Crafts, New York, 1969.

2.

Lasnitzki, I., CELLS AND TISSUES IN CULTURE, Volumel, Editor E. N. Willmer, Academic Press, London, 1965, p. 591.

3.

Durham, N. N., and Leach, K. K., CAN. J. MICROBIOL. 7,

4.

Durham, N. N., and Adams, L. S., BIOCHIM. BIOPHYS. ACTA 121, 90 (1965).

5.

Dorfman, R. I., BIOLOGICAL ACTIVITIES OF STEROIDS IN RELATION TO CANCER, Editors G. Pincus and E. Voller, Academic Press, New York, 1960, p. 445.

6.

Salhanick, H. A., and Berliner, D. L., J. BIOL. CHEM. 227, 583

Appleton-

75 (1961).

(1957 ). 7.

Morgan, J. G.,,Berlin, K. D., Durham, N. N., and Chesnut, R. W., J. ORG. CHEM. 36, 1599 (1971).

8.

Chesnut, R. W., Haslam, D. F., Berlin, K. D., Morgan, J. and Durham, N. N., BACTERIOL. PROC. 7, (197'1).

9*

Gomori, G., STAIN TECHNOL. 25,

10.

81 (1951).

Bray, G. A., ANAL. BIOCHEM. 1, 279 (1960).

11. Robison, G. A., Butcher, R. W., and Sutherland, E. W., ANN. REV. BIOCHEM. 37, 149 (1968).

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12. Johnson, G. S., Friedman, R. M., and Pastan, I., PROC. NAT. ACAD. SCI. 68, 425 (lfll). 13

l

Riggs, T. R., BIOCHEMICAL ACTIONS OF HORMONES, Volume I, Editor G. Litwack, Academic Press, New York, 190, p. 157.

14.

Higgins, M. L., Shaw, T. J., Tillman, M. C. and Leach, F. R., EXPTL. CELL WS. 56, 25 (1969).

15.

Allison, A., THE BIOLOGICAL BASIS OF MEDICINE, Volume 1, Editors E. E. Bittar and N. Bittar, Academic Press, New York, 1968, p. 210.