In vitro Cytotoxicity Testing of Natural Products Using Guinea-pig Ear Keratinocytes

Ann Harris, Alison T. Keene and J. David Phillipson Department of Pharmacognosy, The School of Pharmacy, University of London, 29-39 Brunswick Square, London WClN IAX, UK

David C. Warhurst Department of Medical Protozoology, London School of Hygiene and Tropical Medicine, University of London, Gower Street, London WC1, UK

A simple test is described in which the inhibition of uptake of [G-'H]thymidine into guinea-pig ear keratinocytes in uitro, has been used as an assessment for cytotoxicity of a series of alkaloids. The alkaloids tested include emetine, colchicine, coronaridine, ajmaline, aricine, vincristine, leurosine, quinamine, 3-epiquinamine, quinine, quinidinone, cinchonidine, berberine and atropine.

Keywords: Cytotoxicity; in uitro test; guinea-pig ear keratinocytes; alkaloids.

INTRODUCTION

The search for novel anticancer drugs from natural sources has shown that although numerous plant products give encouraging results in initial biological screening tests, in many instances the activity can be attributed to general cytotoxicity (Suffness and Douros, 1982). Among the classes of natural products which show non-selective cytotoxicity are tannins, alkaloids, diterpenes, sesquiterpenes and saponins (Farnsworth et al., 1975; Schepartz, 1976; Sticher, 1977; Warhurst, 1985). Hence, when plant extracts and isolated natural products are being investigated as potential chemotherapeutic agents, it is essential that at an early stage, specificity of action is distinguished from non-selective toxicity and that there is some estimation of therapeutic index (Connors and Phillips, 1975; White, 1982; Warhurst, 1985). In vitro screens are highly sensitive, low cost, have

a capacity for rapid turnover and can be used as an initial test procedure before resorting to in vivo testing. Mammalian cells fulfil many of the criteria required for in vitro testing and are of particular use for natural product screening because only small quantities of test compound are needed (Perdue, 1982). A number of different mammalian cell lines of ordinary and neoplastic cells taken from different origins and species have been developed allowing adaptable techniques for the assessment of cytotoxi- city (Paul, 1975; Sorg, 1981). Guinea-pig ear keratinocytes (GPKs) are one such type of cell line which has been used for cytotoxicity testing (Riley, 1980) and the methodology outlined in this paper is based on these established techniques.

MATERIALS AND METHODS

Medium and reagents. The following sterile solutions were used: (a) Final growth medium. Minimum essential medium (MEM) modified with Earle's salts and 20 mmol Hepes buffer [Flow Laboratories] 500 mL, foetal calf serum (virus and mycoplasma screened) [Gibco Ltd] 50 mL, L-glutamine (200 nmol/solution [Gibco Ltd] 5 mL, sodium bicarb- onate solution, 7.5% [Gibco Ltd] 2 mL, penicillin and streptomycin solution 1 mL. The solution was stored at +4"C and warmed to 37°C prior to use. (b) Penicillin and streptomycin solution. Penicillin [Sigma Ltd] 50,000 units, streptomycin [Sigma Ltd] 50,000 pg, distilled water to 1 mL. The solution was sterilised by filtration through a 0.22 pm filter and stored in a deep freezer. (c) Trypsin solution. Sodium chloride [BDH Ltd] 0.8 g, potassium chloride [BDH Ltd] 0.04 g, disodium orthophosphate [BDH Ltd] 0.006 g, dextrose [BDH Ltd] 0.1 g, trypsin powder [Hopkins and Williams Ltd], glass distilled water to 100mL. The ingredients were dissolved in the order stated and stirred for 15min with a magnetic stirrer. The resulting solution was centrifuged at +4"C at 10,000rpm for 1 h, the supernatant decanted and sterilised by filtration using a 0.22pm filter. The solution was stored at -18 "C. Before use, 0.3 mL of 7.5% w/v sodium bicarbonate solution was added to 10 mL of the trypsin solution. (d) Dimethylsulphoxide [BDH Ltd] was sterilised by autoclaving. Non-sterile solutions used were (a) Phosphate buffered saline (PBS). One PBS tablet [Oxord Ltd] was dissolved in 100 mL of glass distilled water. (b) Trichloracetic acid (TCA) solution. TCA 5mL, was diluted with glass

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PHMOTHERAPY RESEARCH, VOL. 2, NO. 1, 1988 25

A. HARRIS E T A L .

distilled water to 100 mL. (c) Scintillation cocktail (scintillation grade reagents). BBOT [BDH Ltd] 8 g, naphthalene [BDH Ltd] 160 g, 2-methoxyethanol [BDH Ltd] 800mL, toluene [May and Baker Ltd] 1,200 mL.

Maintenance of cultures. Guinea-pig ear keratinocytes (GPKs) [Flow Laboratories] were grown routinely in vitro as monolayers in Falcon (25 and 75 cm') flasks under aseptic conditions. Cultures were viewed daily by inverted microscope in order to monitor growth and to check for contamination. Healthy cells grew in stellate angular shapes whereas dead or dying cells were rounded. The maintenance of cultures utilised the method of Riley (1980). Cultures were subcul- tured routinely on every 3rd or 4th day. When the cultures were confluent, spent media were removed and trypsin solution added to detach the cells from the flask surface. The detached cells were transferred to sterile centrifuge tubes (10 mL) and spun at 1000 rpm for 2min, the supernatant discarded and the cells resuspended in 1 mL of media and mixed thoroughly. 50pL of resulting suspension was then added to 25mL of normal saline and counted using a Coulter counter. The concentration of the original suspension was then calculated and adjusted to give a final volume with 1 x lo6 cells mL-l. Culture flasks were then seeded to give a concentration of either lo4 or 10' cells mL-' and were incubated at 37°C. Routine autoradiographic screening was carried out on the cultures in order to detect any contamination by mycoplasma (Riley et a l . , 1973).

Cryopreservation of cell Lines. Cells were trypsinised, counted and resuspended in growth media containing 7.5% dimethylsulphoxide as a cryoprotectant. The final concentration of the cells was 2 X lo6 mL-'. The cell suspension was then placed in a 2mL screw top ampoule and left at +4 "C for 3 h before placing in a biological freezer unit and frozen at -70 "C (at a rate of -1" C min-I). After leaving overnight, they were placed and stored in liquid nitrogen. When required for use, cells were removed from liquid nitrogen and rapidly thawed in tepid water. The contents of one ampoule were placed in a Falcon flask with 10mL of growth media, the media changed the following day and the cells subcultured as normal when required.

Cytotoxicity test protocol. Twenty-four well multi-well trays were seeded with GPKs by addition of 1 mL of a 5 x lo4 cell mL-' suspension to each well. The cells were incubated for 48h and used for testing when they were subconfluent. Serial dilutions of the test compound were made in growth medium within the range of 0.01 to 100pgmL-l. The dilutions were made in quadruplicate and added to each well after the spent medium had been removed; control tests without test compound were also carried out at the same time. The cells were incubated at 37 "C for 3 h, then [G-3H]thymidine added to each well to give a final concentration of 10 pCi mL-' and the cells were incubated for a further 30min. After the incubation period, excess medium was removed, each well washed with 1 mLPBS solution and twice with 5 % TCA, the second wash being left for 5min. The two

PBS washes were repeated leaving behind a TCA-insoluble residue containing DNA. The well trays were then dried in a stream of warm air and the residue digested overnight in 250pl of N.NaOH at +4 "C. The level of [G-3H]thymidine remaining in each well was then determined by adding 200pL of the sodium hydroxide digest to 7mL of scintillation cocktail and counting for 1 min in a Packard Tri Carb scintillation counter (Riley, 1980).

Analysis of results. The percentage inhibition (i) calculated for each concentration was transformed into probit values (Finney, 1951). The sigmoid log dose-response curve was transformed to a more nearly linear relationship by using probit of percentage inhibition, but these results were then analysed by regression analysis, rather than the complete probit analysis described by Finney (1951). The 95% confidence interval for the IC5,, values of each test was calculated from the variance of the regression coefficient (Bailey, 1961) and the result converted back to arithmetical values.

RESULTS

The ICso values of a series of isoquinoline, quinoline, indole and tropane alkaloids (1-14) were determined against GPKs by the in vitro test procedure. The results expressed in gmL-' and as mM are presented in Table 1 and they represent the ability of each alkaloid to inhibit the incorporation of [G-3H]- thymidine into the cells as determined in at least two separate experiments. The percentage inhibition values (i) were transformed into probit values (Probability Unit) in order to provide a straight dose response. The results may be expressed graphically by plotting Probit against log concentration and this is illustrated for four of the alkaloids in Fig. 1. Regression analysis was performed in order to find the best straight line through the points obtained. Estimation of the closeness of fit of points to the regression line is given by the correlation coefficient and the significance of this value (probability p that drug concentrations is not related to probit percentage inhibition) is given in Table 1. Experimental results where p > 0.05 were considered unsatisfactory and discarded.

\

1

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IN ViTRO CYTOXICITY TESTING OF NATURAL PRODUCTS

Me0 NHCOCH3

M e o q o \

OMe 2

MeOOC 3

OH

Me00

6 R’zOH, R 2 = H , R3=CH0

7 R1 - R 2 z -0- , R3= Me

8 c-3 H&

9 C - 3 H p

10 R = O M e

11 R = H

Me0

12

13

m)- OC 0 - :H a CH20H

14

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A. HARRIS E T A L . ~~

Table 1. Cytotoxic activity of a series of alkaloids as assessed by the inhibition of inhibition of incorporation of [G-'Hlthymidine into GPK cells in uitro

Alkaloid

Ernetine (1) Colchicine (2) Coronaridine (3) Ajmaline (4) Aricine Vincristine (6) Leurosine (7) Quinamine (8) 3-epiquinarnine (9) Quinine (10) Cinchonidine (11) Quinidinone (12) Berberine (13) Atropine (14)

"50 95% C.I. Correlation P "50 Cytotoxiciw relative ( p g mL-') llrg mL-V coefficient (mM) to emetine (molar basis)

0.02 0.58 4.0

>loo >I00 12.1 5.2

21.6 1 .o

>loo >loo 20.9 14.2 >loo

0.018- 0.022 0.20 - 0.94 1.2 -13.1

- 7.7 -18.3 4.1 - 6.6

13.3 -53.2 0.73 - 1.4

13.8 -31.8 7.5 -27.0

0.9677 0.9393 0.9569

- 0.8976 0.9706 0.9263 0.9574

0.9451 0.8063

0.001 0.001 0.001

0.001 0.001 0.001 0.001

- 0.010 0.010 -

4.2 x 1 0 ~ 1 . 4 ~ 1 0 - ~ 1.2 x 10-2

>0.3 >0.3

1.5 X lo- ' 6.4 x 6.9 X lo-' 3.2 x 10-3

>0.3 >0.3

6.4 x 10' 4.2 X 10'

>0.3

1 33

290

- 357 152

1640 76

1520 1000

DISCUSSION

In order to provide comparative data for the applicability of GPK in vitro cytotoxicity, a number of alkaloids with differing modes of cytotoxic action were investigated. Emetine (l), colchicine (2), coronaridine (3), vincristine (6) and leurosine (7) were chosen as compounds which possess two different modes of cytotoxic action (Grollman, 1967; Hoebeke and Van Nijen, 1975; Suffness and Douros, 1980). The in vitru test used measures the level of inhibition of DNA synthesis (Riley et al. , 1973) and it is not surprising that emetine (1) which acts primarily by inhibition of DNA synthesis and of protein synthesis, proved to be the most potent compound (IC50 0.02 pg mL-'; 4.2 x mM). The toxicities of the other alkaloids to GPK cells have been expressed relative to emetine on a molar basis (Table 1). Colchicine (2) which acts by inhibition of microtubule formation was determined as being 33 times less active than emetine (IGO 0.58 pg mL-'; 1.4 X lop3). The dimeric indole alkal- oids vincristine (6) and leurosine (7) were determined as being much less toxic than emetine (604 and 261 times less active). The in vitro test used is a rapid one

and the short term exposure of the GPK cells to the alkaloids may result in an underestimation of cytotoxicity. Two monomeric indole alkaloids ajmaline (4) and aricine (5) were judged as being inactive against GPK cells (ICs0 values >lo0 pg mL-') whereas the monomeric indole alkal- oid coronaridine (3) was shown to be more potent than either vinblastine or vincristine. Quinamine (8) and epiquinamine (9) showed interesting differences in their cytoxicity with ratios to emetine of 1640 and 76, respectively. Quinine (lo), cinchonidine (11) and atropine (14) were inactive with ICsO values of >lo0 pg mL-l whereas quinidinone (12) showed only very slight activity (IC50 value 20.9 pg mL-'). Berberine (13) which has been used as an amoebicidal and as an antimicrobial drug demonstrated slight activity with an IC50 value of 14.2pgmL-I (1000 times less cytotoxic than emetine).

In designing a screening programme for cytotoxic agents, it should be borne in mind that few in vitro tests allow for a comprehensive study of different modes of cytotoxic action and that more than one test should be utilised. The GPK cell line will detect compounds which inhibit DNA synthesis, primarily or secondarily, during the course of the test whereas these results would suggest that it is relatively insensitive to compounds with different modes of action. The test may be used to determine specificity of biological activity as illustrated by the following example. Quassin, a bitter-testing terpenoid, had an IC50 value of 66pgmL-' in the GPK test whereas when assessed for its activity against Entarnoeba histolyticu in vitru its value was 0.5 pgmL-' (favourable ratio of 132 to 1) (Harris and Phillipson, 1982).

Acknowledgements - 4 - 3 - 2 - 1 0 1 2 3 4

Log concentration

Figure 1. Cytotoxicity of ernetine, colchicine, coronaridine and vincristine to GPK cells in vitro.

We are grateful to Dr P. A . Riley (University College, University of London) for providing the GPK cell line, for his invaluable help with the test protocol and to Dr G. A. Cordell (University of Illinois) for gifts of cytotoxic alkaloids.

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Suffness, M., and Douros, J. D. (1980). Miscellaneous natural products with antitumour activity. In Anticancer Agents Based on Natural Product Models, ed. by M. J. Cassady and J. D. Douros, pp. 465-487. Academic Press, New York.

Suffness, M., and Douros, J. D. (1982). Current status of the NCI plant and animal product programme. J. Nat. Prod. 45,

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1-14.

Received 5 December 1986; accepted (revised) 20 August 1987.

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