Europ. J. Cancer Vcl. 12, pp. 1003-1009. Pergamon Press 1976. Printed in Great Britain

A Screening for Selective Anticancer Agents Among Plant Respiratory Inhibitors*

MARIO GOSALVEZ, R. GARCfA CANERO t and M. BLANCO Bioqufmica Experimental. Clinica Puerta de Hierro. Facultad de Medicina.

Universidad Autdnoma. Madrid. Spain

Abstract--Previous work of our laboratory showed that some useful anticancer agents behave as respiratory inhibitors with some specific activity in tumor cells and that, in some cases, the effect on respiration would represent the mechanism of antitumor action. On this basis, a screening among plant compounds was undertaken to select respiratory inhibitors, selective for tumor cell respiration. Out of thirty compounds, four were found to be potent respiratory inhibitors somewhat specific for tumor cells. Two of these com- pounds appeared with substantial antitumor activity in mice inoculated with L1210. These results would' support the interest of respiratory inhibition as a selective approach to anticancer chemotherapy.

INTRODUCTION

WE REPORTED, previously, that the anticancer agent VM-26 inlhibits NADH linked respiration at low concentrations [1] and that this respira- tory inhibition seemed to be somewhat more specific for certain tumor cells than for certain normal cells, eitlher studying the drug in in vitro or in in vivo respiration [2, 3]. The fact that the respiratory inhibition with VM-26 is obtained well within the range of chemotherapeutic doses, suggested[ that its mechanism of anti- tumor action is associated with its effect on respiration [1-2;]. Respiratory inhibition and respiratory selectivity was also found by our group in other anticancer agents as ellipticene and some triazine and naphtoquinone deriva- tives [2, 4]. All of these studies support our view that an anticancer chemotherapy, based on specific respiratory inhibitors, must be worth investigating [5].

Accepted 14June 1976.

*This work was supported by contract No. NCI-CM- 53792 from the Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, DHEW and by grant No. 12-199-75 from the Spanish Instituto Nacional de Previsi6n.

~'Recipient of a fellowship from the Asociaci6n Espafiola Contra el C~ncer.

In this report, we present a screening for respiratory inhibitors selective for tumor cell respiration, carried out among plant compounds with chemical structure likely to interact with the mitochondrial respiratory chain. Thir ty compounds were selected and studied, measuring their effects in the respiration and oxidative phosphorylation of isolated mito- chondria and tumor and normal cell suspen- sions. Among them, four compounds were found to be potent inhibitors of cell respiration in vitro and in vivo showing some specific activity in tumor cell respiration. Two of these com- pounds appeared with substantial anti tumor activity in mice inoculated with L1210. These results support the interest of respiratory in- hibition as a selective approach to anticancer chemotherapy. Additionally, these results emphasize the interest of biochemical screen- ings aimed at rationally defined specific biochemical targets for andcancer chemo- therapy as a means of detecting potential anticancer agents in high yield.

MATERIAL AND METHODS

Measurement o f respiration and oxidative phospho- rylation in rat liver mitochondria

Rat liver mitochondria were isolated by a modification of the method of Schneider [6].

1003

1004 Mario Gosdlvez, R. Garda Ca~ero and M. Blanco

Mitochondrial respiration (1 mg mitochondrial protein per ml) was measured with glutamate plus malate (5 mM), or succinate (5 mM) as substrate, in the absence (state 4 respiration) or presence (state 3 respiration) of ADP (0.3 mM). ADP:0 ratios were determined as described by Estabrook [7]. The respiration was measured in a Clark-type oxygen electrode (3 ml chamber) in a medium composed of 225 mM sucrose, 20 mM KC1, 7 mM MgC12, 10 mM Tris-HC1 and 5 mM Tris-Pi (pH 7.2, 22°C). The drugs were added in microliter quantities, dissolved in 50/50% dimethyl- formamide and dimethylsulfoxide, or in water, depending on the drug's solubility. Eight minutes of pre-incubation of the drug with the mitochondria were allowed before the deter- mination of the respiration.

Measurements of the respiration of Ehrlich ascites tumor cells

The respiration of Ehrlich ascites tumor cells (20-30 x 106 cells/ml), hypertriploid cells) was determined in the oxygen electrode, in a medium composed of KC1 (6.16 raM), Na2- HPO4 (9.35 mM), NaH2PO4 (1.65 mM) and NaC1 (0.9 %) (pH 7, 22°C) [8]. The respiration rate, after 15 minutes, in the presence of the drug, was compared with the control respiration of non-treated cells. As cells used in these experiments were collected from 6-day-grown Ehrlich ascites tumors (hypertriploid strain), and hemoglobin-free suspensions were prepared as described by Chance and Hess [8].

Measurement of pig leucocyte respiration Mixed populations of leucocytes including

lymphocytes were isolated by differential centrifugation, from the buffy coat of 2 liters of citrated pig blood. The respiration of the cell suspensions was measured in the Clark-type electrode, using the same medium described for the measurement of Ehrlich ascites tumor cell respiration.

Measurement of rat liver cell respiration Anesthetized rats (nembutal) were opened

and the portal vein and superior vena cava were cannulated in order to perfuse the liver with Krebs-Ringer phosphate salt solution (saturated with oxygen and CO2). Once the liver was well-washed, the perfusion was contin- ued with the same solution, supplemented with bovine serum albumin (2"5%), collagenase II (0-05 %) and hyaluronidase (0.1%). Afterwards the liver was isolated from the rat, minced with scissors and incubated at 37°C during 20 min,

with the Krebs enzyme solution, under con- tinuous oxygen gassing. After filtering the cell syspensions with gauze, the isolated liver ceils were centrifugated at 1000 rev/min and were washed twice with the Krebs-Ringer phosphate medium. The liver cell respiration was meas- ured in a Clark-type oxygen electrode in the same assay medium that was used for the Ehrlich tumor ceils.

Measurement of leukemia L1210 cell respiration DBA/2 mice, bearing leukemia L1210 in

ascites form, were received from the National Cancer Institute, and the tumors were trans- planted to mice, resulting from the cross of DBA and C3H mice, by inoculating 105 cells intraperitoneally. After seven days of tumor growth, cells were collected, washed, freed of blood (by a 30 see osmotic shock) and con- centrated. Cell respiration was measured in a Clark-type oxygen electrode, using the same medium that was employed for the Ehrlich ascites tumor cells.

Drugs, solvents, substrates and cofactors A11 plant products were supplied in milligram

quantities by Professor A. Gonz~lez, Instituto de Investigaeiones Quimicas, La Laguna, Tenerife, Spain, a n d almost all were new chromenes and diterpenes, extracted and purified from plants of the Canary Islands. [9-12]. The structure of these compounds is shown in Fig. 1. The chemical names are, respectively: 7H-Furo (3,2-g) (1) benzopyran- 7-one, 6- (1,1-dimethyl-2-propen)- (chalepen- sine) ; 5-benzo(2-(1-methylethylen)-) furan- acrylic acid, lactone (oroselone); 4H, 6H- benzo (1,2b : 5,4-b') dipyran-4-one, 8,8-dimethyl 2-methyl-5-hydroxy-10-(3,3-dimethyl-propen- 2-)- (pulverochromenol) ; 4H, 6H-benzo(1,2b: 5,4-b') dipyran-4-one, 8,8-dimethyl-2-methyl- 5-hydroxy - (3,3 - dimethyl - allyl - 2 - ) - (allyl - spathelia-chromene) ; 6,7-furan-(2,3-dihydro)- chromen-8-ol, 2,3-dimethoxic 5-R- (pitilline)- 5-benzo (2-dimethyl-hydroxymethyl-) furan- acrylic acid, lactone- (oroselol) ; 5,6: 7,8-(2'H, 2'H-dipyran)- chromone, 2-methyl- (spathelia- bis-chromene); 3,4-furan (2,3-dihydro)-ciclo- deca-5,9-diene, 6-hydroximethyl-2- (3,4-hydro- ximethyl - 2 - (3,4- hydroxi- 2 -en) - carboxilate- (cnicine); 7H-furo (3,2-g) (1) benzo pyran-7- one, 4-nitro-7-methoxy-(nitroxantotoxin). The compounds were used as pure products, dissolved in water or 50%/50% dimethyl- formamide and dimethylsylphoxide and were added to mitochondria cells in microliter quantities. Solvents were used as controls.

A Screening for Selective Anticaneer Agents Among Plant Respiratory Inhibitors 1005

Substrates, salts and cofactors were purchased from Sigma Company. Mitochondrial protein was determined by the biuret reaction. Cell concentrations were calculated by cell count in a hemocytometer after vital staining.

RESULTS

Thir ty purified plant compounds were selec- ted from the stock of plant products purified by Professor Gonz~dez at the Instituto of Investi- gaciones Q ufmicas of La Laguna (Tenerife). The compounds were selected by their chemical structure likely to interact with the mito- chondrial respiratory chain and were pre- liminarily tested at 100/aM concentration in isolated mitochondria. Those compounds show- ing at least a five per cent inhibition of mito- chondrial respiration with glutamate plus malate, at such concentration, were accepted in the screening. Table 1 lists the twelve compounds found with such activity and their effects in mitochondria. These twelve com- pounds were titrated in the isolated mito- chondria at increasing concentrations and those showing a 100 % inhibition of mitochondrial respiration at less than 300 pM concentrations, continued in the screening. The first six com- pounds of Table: 1 plus the last compound of the table had such activity. These compounds were titrated in isolated mitochondria with glutamate plus raalate or succinate plus or less ADP, providing thus, the evaluation of its effect on mitochondrial state 4 and state 3 respiration and on ADP: 0 ratio. These com- pounds were also assayed on the respiration of Ehrlich ascites ,cell suspensions to determine the concentration needed for 50 % inhibition. Table 1 shows the effect of the compounds on

mitochondrial respiration with glutamate plus malate or with succinate as substrate in the presence of ADP and the concentration of the compounds for 50 % inhibition of the respira- tion of Ehrlich ascites cells. It can be observed that there is not a close relationship between the potency of the compounds in mitochondria and their potency in the cells. This would be due to differences in cell permeability for each drug. All of the seven more effective compounds except oroselona, inhibited succinate respira- tion, thus behaving as inhibitors of the anti- mycin type. None of them inhibited the mito- chondrial respiration with ascorbate (not shown). Oroselona however, appeared as a pure inhibitor of the rotenone type. Trachino- diol, pulverochromenol and spathelia-bis- chromeno inhibited, partially, mitochondrial ADP:0 ratio and induced a partial activation of mitochondrial respiration in state 4 (not shown), therefore behaving as mixed inhibitors- uncouplers.

Of the seven more active compounds, only trachinodiol, oroselona, chalespensine and pulverochromenol were available in sufficient quantities to be compared in the respiration of tumor and normal cells. Fig. 2 depicts the effect of increasing concentrations of these compounds on the respiration of pig leucocytes, rat liver isolated cells and mice leukemia 1210 and Ehrlich ascites cells. The cell concentration of the four types of cells was 200, 1, 50 and 50 x 106 cells/ml, respectively, as necessary to yield equivalent cell respiration (2) (50 nano- atoms O2/min) with all the cell types. Under these conditions, the known respiratory in- hibitor rotenone has exactly the same potency in the four types of cells and the cells are comparable. Differences of permeability among

Table 1. Effects of plant compounds in mitochondrial and cell respiration

Compound

Inhibition of mitochondrial respiration with Glutamate plus with Succinate Malate

/tM % Inhibition/tM % Inhibition

Inhibition of F, ATC Respiration

BM % Inhibition

Trachinodiol (diacetate) 0'75 50 0.6 50 I00 50 Chalepensine 3.5 50 50.0 50 60 50 Oroselona 7.0 50 100.0 0 90 50 Pulverochromenol 22"0 50 15.0 50 40 50 Epicandieandiol (dlacetate) 25.0 50 15.0 50 30 50 Allyl spatheliachromene 38-0 50 24.0 50 600 50 Pitilline 100.0 40 . . . . Oroselol 100.0 25 . . . . Pristimerine 100.0 20 . . . . Nitroxantotoxine 100.0 10 . . . . Cnieine 100.0 5 . . . . Spathelia-bis-ehrornene I00.0 5 330.0 50 760 50

1006 Mario Gosdlvez, R. Garda Cahero and M. Blanco

Common name Formula

Common name Formula

Trachimodiol diacetafe

Cholepensine

Oroselone

Pulverochromenol

Epicon-dlcon-diol

diocetate

Allykspothelio

chromene

OH

P,,,,,,n. I % c o - v ~ ,

CH 3 Oroselol

O 0

o. c'~x,'=°% H~C I -

"1

o Pr .morio. L 1 1 % H O " ~ ~

CHSo CH3

Spatelio-bls- C~zOH

5 ~ H.C~. CH 3 chromene CH-

a.J i OC _ CHOH_CH20 H

Cnicine OCOCH~,

HO-CH 2 0 " ~ 0

OH 0 NO

O-CH 3

Fig. I. Chemical formula of principal natural compounds used in these studies.

cell types for the different compounds, are matched by the 15 min of pre-incubation of the drug with the cells. Such conditions of equivalent respiration and pre-incubation per- mitted the detection of selective inhibition of Ehrlich ascites cells by VM-26, in Comparison

1.5

0 . 5

1 I

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Fig. 2. Effect of trachinodiol (black circles) oroselone (open circles), chalepensine (triangles) and pulverochromenol (crosses) on the respiration of pig leucocytes, (upper left graph), rat hepatocytes (upper right graph), leukemia 1210 (lower left graph) and Ehrlich ascites cells (lower right graph).

with pig leucocytes [2]. This respiratory selec- tivity was later confirmed in situ and in vivo between two normal and tumor tissues of the same animal [3]. Thus, in a first approxima- tion, the experiment of Fig. 2 permits the definition of specificity of an inhibitor for tumor cell respiration. It is first apparent that oroselona is quite less active in inhibiting the respiration of the leucocytes and hepatocytes than in inhibiting the respiration of the tumor cells. The following compound with high specificity is trachinodiol. Table 2 shows the factor of specificity for each compound in each cell pair [4]. Data on spathelia-bis-chromene is also shown, which is not shown in Fig. 2. The specificity is defined by two numbers, the absolute specificity factor (ASF) and the relative specificity factor (RSF) [4]. ASF is calculated by the ratio between the maximum concentra- tion with no effect on respiration of normal cells and the minimum concentration required for 80 % inhibition of tumor cell respiration. The RSF is calculated by the ratio between the concentration necessary to inhibit 80 % tumor respiration [4] and the concentration necessary to inhibit 80 % normal cell respiration. Oro-

A Screening for Selective Anticancer Agents Among Plant Respiratoo, Inhibitors 1007

Table 2. Specificity of plant compounds for tumor cell respiration

Leucocytes Leucocytes Liver cells Liver cells EATG Leukemia 1210 EATG Leukemia 1210

Compound ASF RSF ASF RSF ASF RSF ASF RSF

Trachinodiol 0"3 2.0 - - - - 7.5 3-3 - - - - (diacetate)

Chalepensine 0-12 1.1 0.15 1.5 0.12 1.1 0.15 1.5 Spathelia-bis- - - 3.3 . . . . . .

chromene Oroselone 3-3 7.1 1.4 1.1 0.66 23-0 0.28 10.0 Pulverochromenol 0-6 2-8 0.55 1.05 0.10 0.65 0.06 0"38

selona appears to be quite specific for the two kinds of tumor cells and shows very high relative specific factors between leucocytes and Ehrlich ascites and between liver and leukemia cells. The selectivity is not so marked between leucocytes and leukemia. Trachinodiol, chale- pensine, pulverochromenol and spathelia-bis- chromene also ,;how specificity in some cell pairs. Chalepensine is the more interesting compound because it shows a good selectivity between leucocytes and leukemia cells. Selec- tivity in this cell pair is difficult to be demon- strated [4] due to the great similitude of both cell types.

Table 3 describes the chemotherapeutic t reatment of L1210 mice with spathelia-bis- chromene, pulverochromenol and chalepensine. Trachinodiol and oroselona were not available in enough quantity for the chemotherapeutic treatments. The: drugs were injected daily starting day one after inoculation of 10 s cells per mice. Spatl)elia-bis-chromene induced a 62 % increase ir~ life span with no survivors after 60 days at the total dose of 7 mg/kg. Higher doses resulted in a short supervivence

due to increased toxicity. Chalepensine, at the total dose of 150 mg/kg managed to yield a 60 % survivors and 70 % increase in life span for the non-surviving mice. These results suggest that both compounds, but above all chalepensine, have substantial chemothera- peutic activity in L1210. L1210 is the first tumor choice of chemotherapeutic screening of the National Cancer Institute in the United States and drugs inducing more than a 50 % increase in life span in this tumor system are considered as potential chemotherapeutic agents [13].

To ascertain if the chemotherapeutic activity of chalepensine was due to its effects in respira- tion, the respiration of L1210 ceils obtained from mice bearing tumors (six days grown), treated at different doses of the drug, was assayed. Fig. 3 shows that at 30 mg/kg of chalepensine, the effective chemotherapeutic dose, there is almost a 100 % inhibition of cell respiration. Essentially similar results are de- picted for pulverochromenol while oroselona appears to have double potency. The results with chalepensine suggest that the mechanism

Table 3. Treatment of L 1210 with plant respiratory inhibitors

Daily dose Total d o s e Increase in Survivors/ Compound (in mg/kg) (in mg/kg) life span treated animals

Spathelia-bis-chromene 1 7 62 % 0]5 3 21 34% 0/5 5 35 28.5% 0/5 8 4o 10% 0/5 10 60 3% 0/5 11 55 3.1% 0/5

Pulverochromenol 8 40 7 % 0/5 10 7o o% o/5

Ghalepensine 8 32 0 % 0/5 lO 60 4% 0/5 30 150 70% 3]5

1008 Mario Gosdlvez, R. Garda Ca~ero and M . Blanco

170

140

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r. 80-

u~ "' 50- iv

• ~ 20

I00

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2 0

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DRUG (~ONCENTRATION

I00 -

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DRUG cONCENTRATION

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DRUG CONCENTRATION DRUG CONCENTRATION

Fig. 3. Respiration of Ehrlich ascites cells isolated from six day grown tumors, two hours after treatment with oroselone (clear circles), chalepensine (triangles) and pulverochromene (crosses) at the indicated doses.

of inhibition of tumor growth is associated with the inhibition of cell respiration. On the other hand, pulverochromenol would have been used in chemotherapeutic t reatment at too low concentrations to cause respiratory inhibition and thus, this fact would have the reason for the lack of chemotherapeutic effect with this drug.

D I S C U S S I O N

Our results have defined chalepensine and spathelia-bis-chromene as having a substantial ant i tumor activity in L1210 mice and therefore these drugs behave as potential anticancer agents. Both compounds were selected out of thirty compounds as being potent respiratory inhibitors with some apparent selectivity for tumor cell respiration. Thus, it would appear that the biochemical screening employed for its selection has a high yield. The definition of the exact yield of the screening awaits the study of oroselona, trachinodiol and pulvero- chromene in L1210, at doses sufficient for complete inhibition of respiration.

The abdominal cavity of mice bearing an ascites tumor might behave in a similar fashion to an in vitro chamber, if the drug is not rapidly

absorbed. Thus, it would be desirable to demonstrate that these drugs inhibit tumor growth injected parenterally. Nevertheless, it is generally accepted that the obtention of surviv- ing animals in intraperitoneally treated tumors reflect a good therapeutic to toxic dose ratio.

The specificity of the compounds for tumor cells respiration was found by comparing nor- mal and tumor cells of different species. The respiration of cell and mitochondria of different species is highly comparable as long as equi- valent cell respiration or similar content of cytochrome is assured [2], but, however, a definite assessment of specificity should include the comparison of normal and tumor tissue of the same animal as has been done for VM-26 [3]. Nevertheless, the good therapeutic activity found with chalepensine would support its selectivity for tumor respiration. As respiratory inhibitors are highly toxic compounds, selec- tivity for tumor cells must be emphasized for them as a class of anticancer agent. The most specific compound found in our screening was oroselona, which has RSF factors from 7 to 23 in various cell pairs. This value means that at least 7 times more drug is necessary to affect the normal cell respiration with the same extent that tumor cell is affected. The existence of such specificity encourages the interest of

A Screening for Selective Anticancer Agents Among Plant Respiratory Inhibitors 1009

respiratory inhibition as a selective approach to anticancer chemotherapy. As activity in L1210 predicts very well potential effects in human tumors [13], the search for high values of RSF in the L1210-1eucocytes pair would be desirable. Our experimental evidence and previous work [2-4] suggest strongly that there is a quantitative difference in sensitivity to some respiratory inhibitors between tumor and normal cells. The reason for such a difference

would possible be based on the recently postu- lated differences of tumor membranes [14-17] or just be considered as a tissue difference, considering tumors in general as a characteristic tissue. The exploiting of such different sensi- tivity with a chemotherapeutic purpose seems appealing and emphasizes the interest of biochemical screenings aimed at rationally defined specific biochemical targets for anti- cancer chemotherapy.

R E F E R E N C E S

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2. M. GOS~LVEZ, M. BLANCO, J. HUNTER, M. MIKO and B. CHANCE, Effects of anticancer agents on the respiration of isolated mitochondria and tumor cells. Europ. J. Cancer 10, 567 (1974).

3. M. GOS~LVEZ, K. GARCfA-CA~ERO and H. REINHOLD, Delayed pyridine nuclcotide reoxidation induced by the anticanccr agent VM-26 as measured in vivo and in situ by NADH microfluorometry. Europ. 3". Cancer ll~ 709 (1975).

4. M. GOS~LVEZ, R. GARCfA-CA~ERO, M. BLANCO and C. GURUCHAm~I-LLOVD, Effects and specificity on the respiration and energy metabolism of tumor cells. Cancer Treat. Rep. 60, 1 (1976).

5. M.J . FIGUERAS and M. GOS.~LVEZ, Inhibition of the growth of Ehrlich ascites tumors by treatment with the respiratory inhibitor Rotenone. Europ. 3". Cancer 9, 529 (1973).

6. W . C . SCHNEIDER, Intracellular distribution of enzymes. III. The oxidation of octanoic acid by rat liver fractions. J. biol. Chem. 176~ 549 (1948).

7. R . W . ESTABROOK, Mitochondrial respiratory control and the polarographic measurement of ADP:O ratios. Methods in Enzymology 10~ 41 (1967).

8. B. CHANC~. and B. HESS, Metabolic control mechanisms. I. Electron transfer in the mammalian cell. 3.. biol. Chem. 234, 2402 (1959).

9. A .G. GONZALEZ, J. P. CASTA~EDA y B. M. Fm~OA, Nuevas cromonas de la neochamelae pulverulenta, ERNDT. An. Quim. 68, 447 (1972).

10. A.G. GONZ~.LEZ, J. L. BRETON, B. M. Fm~OA and J. G. Lms, Constituents of labiatae--IX. Trachinodiol and trachinol, two new diterpenes from Sideritis canariensis Ait. Tetrahedron Lett. 33, 3097 (1971).

11. A . G . GONZALEZ, J. D. MARTIn y C. P~m~z, Nuevas cromonas de la neo- chamalae pulverulenta. An. Quim. 68, 709 (1972).

12. A.G. GONZALEZ. Unpublished data. 13. V. BONO. (Personal communication). Drug Research and Development,

Division of Cancer Treatment, Bethesda, Md. 14. J .C. ARcos, Ultrastructural alterations of the mitochondrial electron transport

chain involving electron leak: Possible basis of "respiratory impairment" in certain tumors. J. theor. Biol. 309 533 (1971).

15. J . C . ARCOS, M. J. TISORS, H. H. GOSCH and J. A. FABIAN, Sequential altera- tions in mitochondrial inner and outer membrane electron transport and in respiratory control during seeding of amino azo-dyes. Stability of phospho- rylation. Conelation with swelling-contraction changes and tumorogenesis threshold. Cancer Res. 29, 1298 (1969).

16. R . E . BArneTT, L. T. FURCHT and R. E. SCOTT, Differences in membrane fluidity and structure in contact inhibited and transferred cells. Proc. nat. Acad. Sci. 71, 1992 (1974).

17. J .L . MARX, Biochemistry of cancer cells: Focus on the cell surface. Science 153~ 1279 (1974).