Cancer Letters, 55 (1990) 209-220
209
Elsevier Scientific Publishers Ireland Ltd.
Solasodine glycosides. In vitro preferential cytotoxicity for human cancer cells B. Dauntera
and
B.E.
Chamb
“The University of Queensland Department ment of Medicine, (Received
of Obstetrics and Gynaecology
Clinical Sciences Building,
Royal Brisbane Hospital,
(Accepted
11 October
12 October
1990)
Introduction
Solamargine
[(ZZR,,?ZR)-spire-5en-3/3-yl-a-
- Zglu)-O-a-L-rhamnoL-rhamnopyanosyl-(1 pytanozyl (I -4glu)-@-D-glucopyranoze],
a
glycoside of solasodine preferentially inhibits the uptake of tritiated thymidine by cancer cells. In contrast,
solamargine
and
at equivalent
the monohave a limited
solasodine of tritiated
thymidine
unstimulated
concentra-
of effect on the uptake
and
diglycosides
for other cell types, inlymphocytes and lym-
phocytes stimulated with Con A. In contrast the solasodine glycosides do not inhibit the uptake of tritiated thymidine with PHA or PWM. thymidine
by lymphocytes The inhibition
uptake by solamargine
and di-glycosides
of solasodine
stimulated of tritiated
and the monoare dependent
upon their cellular uptake by endogenous endocytic lectins (EELS). The mode of action of the
solasodine
glycosides, in particular appears to be the induction of cell
solamargine, lysis,
as
determined
by
morphological
ex-
amination.
Keywords: solasodine glycosides; cytotoxic; endogenous lectins; human cells; in vitro Correspondence
BEC; cancer
to: B., Daunter, Senior Lecturer, The University
of Queensland Department of Obstetrics and Gynecology,
Clinical
Sciences Building, Royal Brisbane Hospital, Herston, Queensland 4006,
Depart-
(Australia)
1990)
Summary
cluding
4006
22 August 1990)
(Revision received
tion,
and bThe University of Queensland
Herston, Queensland
Australia.
0304~3835/90/$03.50
0
Soiasodine glycosides have been isolated and purified as a mixture: solamargine (33%), solasonine (33%) and di- and monoglycosides (34%) [12]. This mixture (BEC) [7] of solasodine glycosides has been shown to be effective in vivo against murine sarcoma 180 [ 101 and human skin cancers [8, Cham, B.E. and Daunter, B., unpublished data, 111. However, these glycosides have not been assessed for their potential use in the treatment of other human cancers or their mode of action established. An in vitro study was therefore undertaken to determine the relative cytotoxic activity and mode of action of the mixture of solasodine glycosides (BEC) compared to other commonly used cytotoxic drugs. In addition, consideration has been given to the mechanism of cellular uptake of the solasodine glycosides. The carbohydrate moieties (glycone) of the solasodine glycosides may interact with endogenous endocytic lectins (EELS) and therefore inhibitory studies with carbohydrates have been undertaken. Interest in plasma membrane endogenous lectins has gained momentum. The first endogenous membrane bound lectin to be identified was the asialoglycoprotein receptor on mammalian hepatocytes with specificity for galactose [1,33]. Interaction of the asialoglycoprotein (ligand) with the receptor results in endocytosis of the complex, the lectin receptor is returned to the cell surface, while the
1990 Elsevier Scientific Publishers Ireland Ltd.
Published and Printed in Ireland
210
ligand is degraded in the lysosome [19,39,42]. Since the discovery of the galactose hepatic endocytic-endogenous-lectin (EEL), other hepatic EELs have been found, for example, for fucose [25] and GalNAc [24]. A number of endogenous lectins, including those for lactose, galactose and glucose have been reported to be expressed during human embryogenesis [20,30] and carcinogenesis [20,30]. These lectins may be involved in cell recognition [30] and substrate binding [23], and have not, as far as we are aware, been demonstrated to be EELS, apart from a fucose EEL on murine leukaemic cells [31] and galactose, maltose and fucose EELS on two human colon carcinoma cell lines [18].
Materials and Methods Uptake of tritiated tbymidine Cells were maintained as monolayers in 1640 tissue culture media (TCM) buffered with Hepes pH 7.5 and supplemented with 10% heat inactivated fetal calf serum (HIFCS): (i) a human ovarian cancer cell line (C180-135) [4], (ii) HeLa cells, (iii) human fibroblasts and (iv) as a cell suspension of lymphoblastoid cells [28] (EBV transformed lymphocytes). Monolayers were trypsinized to form a cell suspension. Cells (2 x 104), replicates of 10, in microtitre plates (Millipore Corp., U.S.A.), were preincubated 7 h, 37.5”C, then 50 ~1 of cytotoxic drug added, pulsed after 21 h for 3 h with 5 &/ml tritiated thymidine. All cells were harvested by vacuum filtration and subjected to &scintillation counting. Similarly, peripheral human blood Iymphocytes were isolated [4] and cultured 2 x 105 lymphocytes in CO, 1640TCM 10% HIFCS. Lymphocytes were stimulated with 20 pg/ml PHA, Con A or PWM, and cultured for 48 h followed by a 24-h pulse [ 131. Inhibition of cytotoxicity was carried out with lactose, lactosyl-albumin, glucose, galactose or rhamnose (Sigma Chemical Co., U.S.A.). Rhamnose, under certain conditions in terms of lectin specificity, can replace galactose [30]. Cell concentrations was 8 x l@/well total volume 250 ~1. The dpm of the experimental replicates were expressed as a percentage of the mean
value of the controls, and the mean value of the experimental replicates calculated (percentage cell survival). The S.D. of the controls did not exceed 10% of their mean value. Cytotoxic drugs All cytotoxic drugs were diluted with HIFCS/TCM. Chlorambucil (Sigma Chemical Co., U.S.A.), l-2 mg in 50 ~1 of DMSO was diluted immediately before use [5]. Cisplatinum, saline solution was obtained from David Bull Laboratories, Australia and vinblastine from Sigma Chemical Co., U.S.A. Solasodine glycosides, 100 mg in 5 ml DMSO was diluted to give a 5% solution of DMSO, and further diluted before use. Appropriate DMSO cytotoxicity studies were also conducted. Solasodine glycosides were supplied as a mixture (BEC) [7] and as separate components, solamargine, solasonine, a mixture of di- and monoglycosides and the aglycone solasodine (heated to 60°C for 1 h DMSO) (Cura Nominees Pty Ltd., Brisbane Australia). - ovarian cancer cells Cells (5 x 104) were transferred (200 PI/chamber of a microscope slide (Lab Tek Miles Scientific). Controls received 50 ~1 HIFCS/TCM and experimental chambers 50 ~1 of solasodine glycosides (BEC) 1.5-3.8 PM/I after 7-h preincubation and incubated for a further 17 h and 3- 15.3 PM/I 21 h preincubation and incubted for a further 3 h. Similarly, the cells were treated with the aglycone solasodine 19.4-96.8 PM/I. The cells were fixed and examined by the Papanicolaou method [36]. Cytology
Results and Dbcumion Uptake of tritiated thymidine Solasonine was ineffective in inhibiting the uptake of tritiated thymidine by ovarian, HeLa, lymphoblastoid and fibroblast cells relative to solamargine (Table I). The mixture of di- and monoglycosides were also ineffective for lymphoblastoid cells and HeLa cells, whereas they caused approximately 30% inhibition for ovarian cancer cells and fibroblasts (Table I). The highest
211 Table
1.
Percentage
S.G. concentration
of tritiated thymidine Percentage
uptake
glycosides.
0.6 ztz 0.1
27.0
0.8
93.0
ztz 8.0
71.0
f
*7
2.6 * 0.5
9.0
FB
LCL
HeLa
7.0 f
of solasodine
survival: cell types
OvCa BEC 12.42 PM/I Solam 11.5 PM/I Solas 11.3 PM/I DMG
(cell survival) in the presence
54*
4
48 *6
35 *
4.2
23 +z 1.7
105.0
f
9.5
117 zt
8.0
96 f
8.3
94.0
f
8.3
97 f
11.0
76 f
5.7
14.45 PM/I S.G. = solasodine glycosides; BEC = mixture, Solam, Solas and DMG; Solam = solamargine; Solas = solasonine; DMG = di- and monoglycosides; OvCa = ovarian cancer cells; HeLa = HeLa cells; LCL = lymphoblastoid cells; FB = fibroblasts.
solamargine. In contrast, solamargine has a limited effect on the uptake of tritiated thymidine by unstimulated lymphocytes and lymphocytes stimulated with Con A, and was ineffective when lymphocytes were stimulated with PHA or PWM. Similarly, solasonine was also found to be ineffective (Table II). The most effective components for inhibiting the uptake of tritiated thymidine by unstimulated lymphocytes and lymphocytes stimulated with Con A were the diand mono-solasodine glycosides, and for
concentration of BEC used contained 6 PM/I of di- and monoglycosides and this would account for lo-12% inhibition for susceptible cells. In order that comparisons may be made with previous studies [8,9, Cham, B.E. and Daunter, B., unpublished data, 111, and because of the limited availability of the individual glycosides, the mixture of solasodine glycosides BEC [7] has been used for further investigations and the molar concentration expressed in terms of the most effective component Table II. Percentage of tritiated thymidine the presence of solasodine glycosides.
S.G.
Percentage
uptake
(cell survival) by the unstimulated
and stimulated
lymphocytes
survival
concentration us
PHA
Con A
PWM 100 l 7.5
BEC
63 zt 7.0
95 *4.0
90 + 8.0
12.42 PM/I Solam
76 zt 9.6
93 f
4.4
79 f
4.0
11.5 ItM/I Solas
97 ZIZ11.8
103 f
5.7
97 f
7.0
110 + 3.8
11.3 FM/I DMG
40 ziz 4.6
93 zt 4.2
57 f
5
103 f
93 f
9.4
9.7
14.45 FM/I n = 10. S.G. = solasodine glycosides; BEC = Solam, Solas and DMG; Solam = solamargine; Solas = solasonine; PHA, Con A and PWM = stimulated lymphocytes. DMG = di- and monoglycosides; US = unstimulated lymphocytes;
in
212
Table III. Dose of cytotoxic (PM/I) to inhibit 50% tritiated thymidine (TRsJ relative to ovarian cancer cells.
Cytot
Solam V.B. C.P. CB
OvCa (PM/I)
HeLa
1.55 9.5 63 60
2.5 2.7 49.5 47.5
LCL
TR,,
uptake (LDJ
TRXI
1.6 0.28 0.78 0.79
3.95 2.6 50 27
as thymidine
FB
ratio
TRSI
(@/I)
(PM/~)
(@I/l)
and expressed
2.5 0.27 0.79 0.45
2.3 0.2 1.5 -
3.65 1.9 95.5’ NSE
Cytot = cytotoxic; Solam = solamargine; V.B. = vinblastine; C.P. = cis-platinum; CB = chlorambucil; Ov CA = ovarian cancer cells; HeLa = HeLa cells; LCL = lymphoblastoid cells; FB = fibroblasts; l = extrapolation; NSE = no significant effect.
reasons given above, BEC has been expressed in terms of their concentration (Table II). From dose-response curves of ovarian cancer cells, HeLa cells, lymphoblastoid cells and fibroblasts, the lethal dose (LD) of cytotoxics required to inhibit 50% (LD,) uptake of tritiated thymidine has been determined. The LD,, of the various cell types has been expressed as a ratio relative to the LD,, of ovarian cancer cells, to give a thymidine uptake ratio (TR,). Therefore TR, values greater than 1.0 indicate that the uptake of tritiated thymidine by ovarian cancer cells is inhibited relative to the other cell type (Table III). From Table III, other TR, can be calculated. The TR,, for fibroblasts/lymphoblastoid cells in the presence of vinblastine
Table IV.
US PHA Con A PWM
LD,
concentration
of cytotoxic
is 0.73, which demonstrates its inhibitory effects on normal cells [14]. Similarly, chlorambucil (alkylating) and cisplatinum (DNA binding) which are used in the treatment of chronic lymphatic leukemia (CLL) and ovarian cancer also have attendant toxicity respectively, [2,14,22], although that of chlorambucil is not reflected by fibroblasts. However, in the case of vinblastine or &-platinum, the low TR, for fibroblasts relative to ovarian cancer cells is evident (Table III). In contrast, solamargine gives a TR,, greater than 2 for fibroblasts and lymphoblastoid cells relative to ovarian cancer cells. The specificity of solamargine for ovarian cancer cells is also reflected by the TR, relative to HeLa cells (Table III). In addition, the molar con-
drugs for unstimulated
and stimulated
lymphocytes.
CB
CP
VB
DMG
(FM/~)
(PM/I)
(cM/l)
PM/~)
33.0 24.6 33.0 22.0
21.0 17.0 11.0 9.0
11.0 19.2t 7.8tt 10.8tt
6.4 14.8’ 11.2” 196.0’ ’
US = unstimulated lymphocytes; PHA, Con A and PWM = stimulated lymphocytes; platinum; VB = vinblastine; DMG = di- monoglycosides of solasodine. ‘Extrapolation r = -0.86 P 0.02 (from Fig. la). ’ *Extrapolation r = -0.91 P = 0.01 (from Fig. la). l l ‘Extrapolation r = -0.89 P O.Ol (from Fig. la). tExtrapolation r = -0.811 P = 0.05 (from Fig. lc). ttExtrapolation r = -0.94 0.001 < P ~0.01 (from Fig. lc).
CB = chlorambucil;
l
CP = cis-
213 Table V. Effect of solamargine: percentage of tritiated thymidine uptake (cell survival) in the presence and absence of lactose, galactose or rhamnose. Cells
Solamargine Cont.
Percentage survival
(rM/l)
No Carbohydrate
OvCa HeLa
1.54 2.4
LCL Fb
3.1 3.1
62 25 24 34 58
zt zt * f i
7 2.6 4 3 9.6
Lactose (1.1 PM/I)
(9) (8) (8) (10) (7)
75 l ll* 24 f 2.9
Galactose (1.1 PM/I)
(7) (10)
47 zt 4.8”‘(10) 100 f 11”’ (7)
Rhamnose (1.1 PM/I)
79 l lO” 24 zt 2.2
(10) (10)
42 zt 3”’ 101 f 10”’
(10) (10)
67 zrz7.5 26 zt 2.1 36 zt 4.5 87 ztz6.7”’
(9) (7) (10) (10)
n = 10. OvCa = Ovarian cancer cell line; HeLa = HeLa cells; LCL = lymphoblastoid cells; Fb = fibroblasts. Significant inhibition of solamargine: l0.01 < P < 0.02. l . P = 0.001. l l*P < 0.001.
centration of solamargine that is required to achieve an LD, for ovarian cancer cells is 6-40 times less than that of the other cytotoxits investigated (Table III). The LD,, from dose-response curves for lymphocytes have been calculated and are presented in Table IV. The LD, for DMG indicates that they are equivalent or more inhibitory for the uptake of tritiated thymidine than other anti-neoplastic drugs investigated, except in the case of PWM-stimulated lymphocytes. The LD,, concentration for the di- and monoglycosides of solasodine (DMG) is increased with stimulated lymphocytes, in particular with PWM stimulation. Stimulated lymphocytes undergo a number of changes, which includes an increase in plasma membrane permeability [26,27,34,37], and this may account for the variable LD, for some of the cytotoxic drugs investigated. However, this situation does not appear to apply to DMG. Firstly, PHA and PWM stimulation of lymphocytes decreases rather than enhancing the effect of DMG. Similarly, this applies to the effect of solamargine and the absence of any effect of solasonine on unstimulated and stimulated lymphocytes (Table II). Secondly, the only difference between the glycosides are their carbohydrate moieties (glycone) , because they
all contain suggests glycosides due to the dogenous applies to Inhibition
the same aglycone solasodine. This that the different effects of the on unstimulated lymphocytes may be presence or absence of different enendocytic lectins (EELS) and this also the other cell types investigated.
of solasodine glycosides The effect of solamargine on HeLa cells is not inhibited by any of the carbohydrates (Table V) . In addition, solasonine which has a carbohydrate moiety (glycone) Glu(l--4)Ga1(2l)Gal, the di- and monoglycosides [Gal( l-4)Glu,Glu(2- 1)Gal Glu(l-4)Gal, Gal(2- l)Gal, Gal and Glu] and the aglycone solasodine do not inhibit the uptake of tritiated thymidine by HeLa cells (Table I, Fig. 1). This demonstrates that HeLa cells express an EEL for the glycone of solamargine [Gal( l -4)Glu(2l)Gal]. In contrast, the effect of solamargine and DMG (present in BEC) on ovarian cancer cells (Table I) is partially inhibited by lactose (Table V) and lactosyl-albumin (Fig. 1). The lactosyl-albumin inhibitory effect is aproximately 4 times that of lactose at equivalent lactose concentration (Fig. 1). This is because glycoconjugates have an increased affinity for their corresponding lectin relative to unconjugated carbohydrates [32].
214
bed
100
90 80
i
10 0
/0 suhival
60 so 40
UM / L
solamargine/solasodino
Fis. 1. Inhibition of solasodine glycosides (BEC mixture expressed in terms of solamargine) cytotoxicity (uptake of tritiated thymidine) by lactose and lactosyl-albumin. a / ovarian cancer cell line; aI / ovarian cancer cell line in the presence of lactose 1.1 PM/I; a2 / ovarian cancer cell line in the presence of factosylalbumin 1.1 FM/I lactose. Top curve / effect of aglycone solasodine; b / Hela cells; c / lymphoblastoid cells; d / fibroblasts.
100
bed
90 80, 70,
x survival
a
60, so, 40, 30, 20, 10.
s
10 uM
1s
/L
20
2s
* 30
sol8sodine
F&. 2. Effect of high doses of solasodine (aglycone) on the uptake of tritiated thymidine by: a / ovarian cancer cell line; b / HeLa cells; c / lymphoblastoid cells; d / fibroblasts.
215
b
d a
uM/L Fig. 3.
sola
sodine
Effect of high doses of solasodine
lymphocytes.
a / unstimulated;
(aglycone) on the uptake of tritiated thymidine b / PHA; c / Con A; d / PWM.
Similarly, lactose and galactose cause partial inhibition of BEC activity for lymphoblastoid cells as well as ovarian cancer cells (Table V), whereas solasonine and the aglycone are not effective (Table I, Fig. 5). This suggests that ovarian cancer cells and lymphoblastoid express EELS for the glycone of solamargine and also EELs for lactose and galactose (Tables I and V) . The EELS for lactose and galactose may also recognize their respective carbohydrate moieties on the solamargine glycone [lactose = Gal(l-4)GIu]. Similarly, this also applies to fibroblasts for which rhamnose is also inhibitory (Table V). Although rhamnose is not found in mammalian glycoconjugates, it can substitute for fl-galactose under certain conditions [30]. The aglycone solasodine does not inhibit the uptake of tritiated thymidine at equivalent concentrations of solamargine (Fig. 1). However, at higher concentrations there is an inhibitory effect, but this is more apparent with ovarian cancer cells (Fig. 2). This may be explained by
by unstimulated
and stimulated
the changes in membrane permeability of cancer cells [16]. It is possible that solasodine, which is a very hydrophobic molecule, undergoes enhanced protein-binding and thus reduces its bioavailability. The inhibitory effects therefore become apparent at higher concentrations. Similarly, in the case of lymphocytes, the aglycone solasodine does not exert an inhibitory effect at equivalent concentrations of DMG. However, at much higher concentrations, there is a substantial effect on unstimulated lymphocytes, which is significantly reduced in the case of stimulated lymphocytes (Fig. 3). This suggests that membrane permeability to the aglycone is decreased in stimulated lymphocytes. However, this would not account for the differential effect of the glycosides on unstimulated and stimulated lymphocytes (Table II) and inhibition by carbohydrates (Table VI). Both glucose and rhamnose inhibit the action of DMG with Con A stimulated lymphocytes, but not unstimulated lymphocytes. This suggests
216
Table VI. Percentage of tritiated thymidine uptake (cell survival) of unstimulated in the presence of solasodine glycosides and carbohydrates.
US Con A
lymphocytes
DMG Cont.
Percentage
WW
No carbohydrate
Lactose (1.1 /WI)
Galactose (1.1 /WI)
Glucose (1.1 ctM/I)
Rhamnose (1.1 sM/l)
4.81 4.81
72 zt 12 85 EIZ11
69 ZIZ12 75 f 5.5
77 f 8.6 84 ZIE11.8
76 zt 15 109 l 15’
85 f 102 f
n = 7. l0.01 < P > 0.001. l ‘0.02 < P > 0.01. DMG = di- and monoglycosides;
Survival
US = unstimulated;
Ovarian cancer cells in the presence
12 11”
Con A = stimulated.
that unstimulated lymphocytes may express EELS for the carbohydrate moieties of the diglycosides Glu(2- 1)Gal and Glu(l-4)Gal, but not Gal( l -4)Glu (lactose), since lactose was not inhibitory (Table VI). Therefore, the inhibition of DMG by glucose and rhamnose for Con A
Fig. 4.
and Con A-stimulated
of the aglycone
stimulated lymphocytes suggests that Con A stimulation results in the expression of additional EELS for glucose and galactose (Table VI). It is established that Con A stimulation gives rise to a subpopulation of T suppressor cells (TS) that inhibit helper T cells (TH) within the total popuia-
solasodine
96.8 PM/I 3 h
(X 1000).
217
Fig. 5.
Ovarian cancer cells in the presence
of solasodine
glycosides
expressed
in terms of solamargine
6.1 FM/I 3
h (x1000).
tion [17]. The TS cells produce soluble factors that inhibit TH cell functions [Zl, 15,371, but these suppressor factors can be inhibited by IVacetyl-glucosamine or rhamnose by complexing the TH cell receptors [3,X]. Therefore, one of the soluble TS cell factors and/or Con A may be involved in receptor (EEL) induction on TH cells. In addition, since solamargine has a limited inhibitory effect on the uptake of tritiated thymidine by unstimulated lymphocytes (Table II), they may also express an EEL for Gal(l-4)Glu(Z1)Gal. Cytology
The aglycone solasodine did not have any observable effect on the ovarian cancer cells at any of the concentrations investigated (Fig. 4). In contrast, with increasing concentration of the solasodine glycosides (expressed as concentration of solamargine) over 3 h, the cytoplasm of the cancer cells undergo dissolution, the nuclei contract and become dark staining (Fig. 5),
nuclei then enlarge, the chromatin clumps, and finally the nuclei disintegrate (Fig. 6). It therefore appears that the inhibition of thymidine uptake by solamargine is the result of cell lysis, which is dependent on the uptake of solamargine by EELS.
Neoglycoprotein conjugates of cytotoxic drugs have been demonstrated to be suitable for the targeting of cytotoxic drugs via endogenous endocytic lectins (EELS) [l&30-32]. However, these studies have used monosaccharide or disaccharide conjugates for drug targeting of EELS which are also expressed by various normal cells [24,25,35]. In contrast, we have demonstrated the existence of a more complex EEL on an ovarian cancer cell line and HeLa cells for the trisaccharide Gal(l--4)Glu(2l)Gal. The ovarian cancer cells also express EELS for lactose and galactose, as demonstrated by
218
Fig. 6.
Ovarian cancer
cells in the presence
of solasodine
glycosides
expressed
in terms of solamagine
15.3 PM, ‘I 3
h (x 1575)
the limited cytotoxicity of the mono- and diglycosides of solasodine and the partial inhibition of BEC cytotoxicity. Nevertheless, it is the expression of the EEL for the trisaccharide [Gal(l-4)Glu(2l)Gal] of the solasodine glycoside solamargine which appears mainly responsible for the increased cytotoxicity of BEC . This is the first time that an EEL for a trisaccharide has been demonstrated on cancer cells relative to normal cells such that the difference in EEL expression may be exploited for increased specific targeting of cytotoxic glycoconjugates. the natural glycosides of In this respect, solasodine offers a starting point for the investigation of EELS on other types of cancer cells. Also, the lytic action of the solasodine glycosides offers the potential for further investigations for their use in the treatment of cancer.
That lymphocyte EELs can be modulated also offers the potential for therapeutic intervention in lymphatic disorders. This has been considered by Monsigny et al. [29] in the case of murine leukemia, by the use of glycosylated albumin conjugated to methotrexate. In addition, it may be possible to regulate the immune response by non-toxic neoglycoconjugates, such that helper and suppressor activities may be modulated. Ackaowiedgements We wish to thank Mr. T Hurst and Mrs. B Sanderson for their technical assistance. Referencea 1
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Gabius, H.J., Engelhardt, R., Hellmann, T., Idoux, P., Monsigny, M., Nagel, G.A. and Vehmeyer, K. (1987) Characterization of membrane lectins in human colon carcinoma cells by flow cytofluorometry, drug targeting and affinity chromatography. Anti Cancer Res., 7, 109-112. Golstein, J.L., Brown, M.S., Anderson, R.G.W., Russell, D.W. and Schneider, W.J. (1985) Receptor mediated endocytotis: Concepts emerging from LDL receptor system. Ann. Rev. Cell Biol., 1, l-39. Grabel, L.B. (1984) Isolation of a putative cell adhesion mediating lectin from teratocarcinoma cells and its possible role in differentiation. J. Cell Dtfferentiation, 15, 121-124. Greene, W.C., Fleisher, T.A. and Waldman T.A. (1981) Soluble suppressor supematants elaborated by concanavalin A activated human mononuclear cells. 1. Characterization of a soluble suppressor T cell proliferation. J. Immunol., 126, 1185-1191. Helson, L., Okonkow, E., Anton, L. and Cvitkovic. E. (1978) Cis-platinum ototoxicity. Clin. Toxicol., 13, 469-471. Hinek, A., Wrenn, D.S. , Mecham, R.P. and Barondes, S.H. (1988) The elasttn receptor A galactoside-binding protein. Science, 239, 1539-1541. Kolb-Bachofen, V., Schlepper-Schafer. J., Hulsmann, D. and Kolb, H. (1984) Gal/NAG/Gal-specific rat liver lectinstheir role in cellular recognition. Biol. Cell., 51, 219-226. Lehrman, M.A., Piio, S.V., Imber, M.J. and Hill, R.L. (1986) The binding of fucose-containing glycoproteins by hepatic lectins. J. Biol. Chem., 261 (16), 7412-7418. Lis, H. and Sharon, N. (1977) In: The Antigens, pp. 4, 429-529. Editor: M. Sela, Academic Press. Lis, H and Sharon, N. (1986). In: The Lectins: Properties, Functions and Applications in Biology and Medicine, pp. 270-291. Editors: 1. Eliener, N. Sharon and I.J. Goldstein, Academic Press Inc. Maynard, D., Musk, P., Daunter, B., Khoo. S.K. and Parsons, P G (1985) Melphalan-resistant lymphoblastic cell lines established from patients with ovarian cancer treated with cross-linking agents. J. Exp. Med. Sci., 63. 333-341. Miller, D.K., Griffiths, E., Lenard, J., and Firestone, R.A. (1983) Cell killing by lysosomotropic detergents. J. Cell. Biol., 97, 1941-1951.
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complex.
J. Cell.
Biol.,
98,
209
Elsevier Scientific Publishers Ireland Ltd.
Solasodine glycosides. In vitro preferential cytotoxicity for human cancer cells B. Dauntera
and
B.E.
Chamb
“The University of Queensland Department ment of Medicine, (Received
of Obstetrics and Gynaecology
Clinical Sciences Building,
Royal Brisbane Hospital,
(Accepted
11 October
12 October
1990)
Introduction
Solamargine
[(ZZR,,?ZR)-spire-5en-3/3-yl-a-
- Zglu)-O-a-L-rhamnoL-rhamnopyanosyl-(1 pytanozyl (I -4glu)-@-D-glucopyranoze],
a
glycoside of solasodine preferentially inhibits the uptake of tritiated thymidine by cancer cells. In contrast,
solamargine
and
at equivalent
the monohave a limited
solasodine of tritiated
thymidine
unstimulated
concentra-
of effect on the uptake
and
diglycosides
for other cell types, inlymphocytes and lym-
phocytes stimulated with Con A. In contrast the solasodine glycosides do not inhibit the uptake of tritiated thymidine with PHA or PWM. thymidine
by lymphocytes The inhibition
uptake by solamargine
and di-glycosides
of solasodine
stimulated of tritiated
and the monoare dependent
upon their cellular uptake by endogenous endocytic lectins (EELS). The mode of action of the
solasodine
glycosides, in particular appears to be the induction of cell
solamargine, lysis,
as
determined
by
morphological
ex-
amination.
Keywords: solasodine glycosides; cytotoxic; endogenous lectins; human cells; in vitro Correspondence
BEC; cancer
to: B., Daunter, Senior Lecturer, The University
of Queensland Department of Obstetrics and Gynecology,
Clinical
Sciences Building, Royal Brisbane Hospital, Herston, Queensland 4006,
Depart-
(Australia)
1990)
Summary
cluding
4006
22 August 1990)
(Revision received
tion,
and bThe University of Queensland
Herston, Queensland
Australia.
0304~3835/90/$03.50
0
Soiasodine glycosides have been isolated and purified as a mixture: solamargine (33%), solasonine (33%) and di- and monoglycosides (34%) [12]. This mixture (BEC) [7] of solasodine glycosides has been shown to be effective in vivo against murine sarcoma 180 [ 101 and human skin cancers [8, Cham, B.E. and Daunter, B., unpublished data, 111. However, these glycosides have not been assessed for their potential use in the treatment of other human cancers or their mode of action established. An in vitro study was therefore undertaken to determine the relative cytotoxic activity and mode of action of the mixture of solasodine glycosides (BEC) compared to other commonly used cytotoxic drugs. In addition, consideration has been given to the mechanism of cellular uptake of the solasodine glycosides. The carbohydrate moieties (glycone) of the solasodine glycosides may interact with endogenous endocytic lectins (EELS) and therefore inhibitory studies with carbohydrates have been undertaken. Interest in plasma membrane endogenous lectins has gained momentum. The first endogenous membrane bound lectin to be identified was the asialoglycoprotein receptor on mammalian hepatocytes with specificity for galactose [1,33]. Interaction of the asialoglycoprotein (ligand) with the receptor results in endocytosis of the complex, the lectin receptor is returned to the cell surface, while the
1990 Elsevier Scientific Publishers Ireland Ltd.
Published and Printed in Ireland
210
ligand is degraded in the lysosome [19,39,42]. Since the discovery of the galactose hepatic endocytic-endogenous-lectin (EEL), other hepatic EELs have been found, for example, for fucose [25] and GalNAc [24]. A number of endogenous lectins, including those for lactose, galactose and glucose have been reported to be expressed during human embryogenesis [20,30] and carcinogenesis [20,30]. These lectins may be involved in cell recognition [30] and substrate binding [23], and have not, as far as we are aware, been demonstrated to be EELS, apart from a fucose EEL on murine leukaemic cells [31] and galactose, maltose and fucose EELS on two human colon carcinoma cell lines [18].
Materials and Methods Uptake of tritiated tbymidine Cells were maintained as monolayers in 1640 tissue culture media (TCM) buffered with Hepes pH 7.5 and supplemented with 10% heat inactivated fetal calf serum (HIFCS): (i) a human ovarian cancer cell line (C180-135) [4], (ii) HeLa cells, (iii) human fibroblasts and (iv) as a cell suspension of lymphoblastoid cells [28] (EBV transformed lymphocytes). Monolayers were trypsinized to form a cell suspension. Cells (2 x 104), replicates of 10, in microtitre plates (Millipore Corp., U.S.A.), were preincubated 7 h, 37.5”C, then 50 ~1 of cytotoxic drug added, pulsed after 21 h for 3 h with 5 &/ml tritiated thymidine. All cells were harvested by vacuum filtration and subjected to &scintillation counting. Similarly, peripheral human blood Iymphocytes were isolated [4] and cultured 2 x 105 lymphocytes in CO, 1640TCM 10% HIFCS. Lymphocytes were stimulated with 20 pg/ml PHA, Con A or PWM, and cultured for 48 h followed by a 24-h pulse [ 131. Inhibition of cytotoxicity was carried out with lactose, lactosyl-albumin, glucose, galactose or rhamnose (Sigma Chemical Co., U.S.A.). Rhamnose, under certain conditions in terms of lectin specificity, can replace galactose [30]. Cell concentrations was 8 x l@/well total volume 250 ~1. The dpm of the experimental replicates were expressed as a percentage of the mean
value of the controls, and the mean value of the experimental replicates calculated (percentage cell survival). The S.D. of the controls did not exceed 10% of their mean value. Cytotoxic drugs All cytotoxic drugs were diluted with HIFCS/TCM. Chlorambucil (Sigma Chemical Co., U.S.A.), l-2 mg in 50 ~1 of DMSO was diluted immediately before use [5]. Cisplatinum, saline solution was obtained from David Bull Laboratories, Australia and vinblastine from Sigma Chemical Co., U.S.A. Solasodine glycosides, 100 mg in 5 ml DMSO was diluted to give a 5% solution of DMSO, and further diluted before use. Appropriate DMSO cytotoxicity studies were also conducted. Solasodine glycosides were supplied as a mixture (BEC) [7] and as separate components, solamargine, solasonine, a mixture of di- and monoglycosides and the aglycone solasodine (heated to 60°C for 1 h DMSO) (Cura Nominees Pty Ltd., Brisbane Australia). - ovarian cancer cells Cells (5 x 104) were transferred (200 PI/chamber of a microscope slide (Lab Tek Miles Scientific). Controls received 50 ~1 HIFCS/TCM and experimental chambers 50 ~1 of solasodine glycosides (BEC) 1.5-3.8 PM/I after 7-h preincubation and incubated for a further 17 h and 3- 15.3 PM/I 21 h preincubation and incubted for a further 3 h. Similarly, the cells were treated with the aglycone solasodine 19.4-96.8 PM/I. The cells were fixed and examined by the Papanicolaou method [36]. Cytology
Results and Dbcumion Uptake of tritiated thymidine Solasonine was ineffective in inhibiting the uptake of tritiated thymidine by ovarian, HeLa, lymphoblastoid and fibroblast cells relative to solamargine (Table I). The mixture of di- and monoglycosides were also ineffective for lymphoblastoid cells and HeLa cells, whereas they caused approximately 30% inhibition for ovarian cancer cells and fibroblasts (Table I). The highest
211 Table
1.
Percentage
S.G. concentration
of tritiated thymidine Percentage
uptake
glycosides.
0.6 ztz 0.1
27.0
0.8
93.0
ztz 8.0
71.0
f
*7
2.6 * 0.5
9.0
FB
LCL
HeLa
7.0 f
of solasodine
survival: cell types
OvCa BEC 12.42 PM/I Solam 11.5 PM/I Solas 11.3 PM/I DMG
(cell survival) in the presence
54*
4
48 *6
35 *
4.2
23 +z 1.7
105.0
f
9.5
117 zt
8.0
96 f
8.3
94.0
f
8.3
97 f
11.0
76 f
5.7
14.45 PM/I S.G. = solasodine glycosides; BEC = mixture, Solam, Solas and DMG; Solam = solamargine; Solas = solasonine; DMG = di- and monoglycosides; OvCa = ovarian cancer cells; HeLa = HeLa cells; LCL = lymphoblastoid cells; FB = fibroblasts.
solamargine. In contrast, solamargine has a limited effect on the uptake of tritiated thymidine by unstimulated lymphocytes and lymphocytes stimulated with Con A, and was ineffective when lymphocytes were stimulated with PHA or PWM. Similarly, solasonine was also found to be ineffective (Table II). The most effective components for inhibiting the uptake of tritiated thymidine by unstimulated lymphocytes and lymphocytes stimulated with Con A were the diand mono-solasodine glycosides, and for
concentration of BEC used contained 6 PM/I of di- and monoglycosides and this would account for lo-12% inhibition for susceptible cells. In order that comparisons may be made with previous studies [8,9, Cham, B.E. and Daunter, B., unpublished data, 111, and because of the limited availability of the individual glycosides, the mixture of solasodine glycosides BEC [7] has been used for further investigations and the molar concentration expressed in terms of the most effective component Table II. Percentage of tritiated thymidine the presence of solasodine glycosides.
S.G.
Percentage
uptake
(cell survival) by the unstimulated
and stimulated
lymphocytes
survival
concentration us
PHA
Con A
PWM 100 l 7.5
BEC
63 zt 7.0
95 *4.0
90 + 8.0
12.42 PM/I Solam
76 zt 9.6
93 f
4.4
79 f
4.0
11.5 ItM/I Solas
97 ZIZ11.8
103 f
5.7
97 f
7.0
110 + 3.8
11.3 FM/I DMG
40 ziz 4.6
93 zt 4.2
57 f
5
103 f
93 f
9.4
9.7
14.45 FM/I n = 10. S.G. = solasodine glycosides; BEC = Solam, Solas and DMG; Solam = solamargine; Solas = solasonine; PHA, Con A and PWM = stimulated lymphocytes. DMG = di- and monoglycosides; US = unstimulated lymphocytes;
in
212
Table III. Dose of cytotoxic (PM/I) to inhibit 50% tritiated thymidine (TRsJ relative to ovarian cancer cells.
Cytot
Solam V.B. C.P. CB
OvCa (PM/I)
HeLa
1.55 9.5 63 60
2.5 2.7 49.5 47.5
LCL
TR,,
uptake (LDJ
TRXI
1.6 0.28 0.78 0.79
3.95 2.6 50 27
as thymidine
FB
ratio
TRSI
(@/I)
(PM/~)
(@I/l)
and expressed
2.5 0.27 0.79 0.45
2.3 0.2 1.5 -
3.65 1.9 95.5’ NSE
Cytot = cytotoxic; Solam = solamargine; V.B. = vinblastine; C.P. = cis-platinum; CB = chlorambucil; Ov CA = ovarian cancer cells; HeLa = HeLa cells; LCL = lymphoblastoid cells; FB = fibroblasts; l = extrapolation; NSE = no significant effect.
reasons given above, BEC has been expressed in terms of their concentration (Table II). From dose-response curves of ovarian cancer cells, HeLa cells, lymphoblastoid cells and fibroblasts, the lethal dose (LD) of cytotoxics required to inhibit 50% (LD,) uptake of tritiated thymidine has been determined. The LD,, of the various cell types has been expressed as a ratio relative to the LD,, of ovarian cancer cells, to give a thymidine uptake ratio (TR,). Therefore TR, values greater than 1.0 indicate that the uptake of tritiated thymidine by ovarian cancer cells is inhibited relative to the other cell type (Table III). From Table III, other TR, can be calculated. The TR,, for fibroblasts/lymphoblastoid cells in the presence of vinblastine
Table IV.
US PHA Con A PWM
LD,
concentration
of cytotoxic
is 0.73, which demonstrates its inhibitory effects on normal cells [14]. Similarly, chlorambucil (alkylating) and cisplatinum (DNA binding) which are used in the treatment of chronic lymphatic leukemia (CLL) and ovarian cancer also have attendant toxicity respectively, [2,14,22], although that of chlorambucil is not reflected by fibroblasts. However, in the case of vinblastine or &-platinum, the low TR, for fibroblasts relative to ovarian cancer cells is evident (Table III). In contrast, solamargine gives a TR,, greater than 2 for fibroblasts and lymphoblastoid cells relative to ovarian cancer cells. The specificity of solamargine for ovarian cancer cells is also reflected by the TR, relative to HeLa cells (Table III). In addition, the molar con-
drugs for unstimulated
and stimulated
lymphocytes.
CB
CP
VB
DMG
(FM/~)
(PM/I)
(cM/l)
PM/~)
33.0 24.6 33.0 22.0
21.0 17.0 11.0 9.0
11.0 19.2t 7.8tt 10.8tt
6.4 14.8’ 11.2” 196.0’ ’
US = unstimulated lymphocytes; PHA, Con A and PWM = stimulated lymphocytes; platinum; VB = vinblastine; DMG = di- monoglycosides of solasodine. ‘Extrapolation r = -0.86 P 0.02 (from Fig. la). ’ *Extrapolation r = -0.91 P = 0.01 (from Fig. la). l l ‘Extrapolation r = -0.89 P O.Ol (from Fig. la). tExtrapolation r = -0.811 P = 0.05 (from Fig. lc). ttExtrapolation r = -0.94 0.001 < P ~0.01 (from Fig. lc).
CB = chlorambucil;
l
CP = cis-
213 Table V. Effect of solamargine: percentage of tritiated thymidine uptake (cell survival) in the presence and absence of lactose, galactose or rhamnose. Cells
Solamargine Cont.
Percentage survival
(rM/l)
No Carbohydrate
OvCa HeLa
1.54 2.4
LCL Fb
3.1 3.1
62 25 24 34 58
zt zt * f i
7 2.6 4 3 9.6
Lactose (1.1 PM/I)
(9) (8) (8) (10) (7)
75 l ll* 24 f 2.9
Galactose (1.1 PM/I)
(7) (10)
47 zt 4.8”‘(10) 100 f 11”’ (7)
Rhamnose (1.1 PM/I)
79 l lO” 24 zt 2.2
(10) (10)
42 zt 3”’ 101 f 10”’
(10) (10)
67 zrz7.5 26 zt 2.1 36 zt 4.5 87 ztz6.7”’
(9) (7) (10) (10)
n = 10. OvCa = Ovarian cancer cell line; HeLa = HeLa cells; LCL = lymphoblastoid cells; Fb = fibroblasts. Significant inhibition of solamargine: l0.01 < P < 0.02. l . P = 0.001. l l*P < 0.001.
centration of solamargine that is required to achieve an LD, for ovarian cancer cells is 6-40 times less than that of the other cytotoxits investigated (Table III). The LD,, from dose-response curves for lymphocytes have been calculated and are presented in Table IV. The LD, for DMG indicates that they are equivalent or more inhibitory for the uptake of tritiated thymidine than other anti-neoplastic drugs investigated, except in the case of PWM-stimulated lymphocytes. The LD,, concentration for the di- and monoglycosides of solasodine (DMG) is increased with stimulated lymphocytes, in particular with PWM stimulation. Stimulated lymphocytes undergo a number of changes, which includes an increase in plasma membrane permeability [26,27,34,37], and this may account for the variable LD, for some of the cytotoxic drugs investigated. However, this situation does not appear to apply to DMG. Firstly, PHA and PWM stimulation of lymphocytes decreases rather than enhancing the effect of DMG. Similarly, this applies to the effect of solamargine and the absence of any effect of solasonine on unstimulated and stimulated lymphocytes (Table II). Secondly, the only difference between the glycosides are their carbohydrate moieties (glycone) , because they
all contain suggests glycosides due to the dogenous applies to Inhibition
the same aglycone solasodine. This that the different effects of the on unstimulated lymphocytes may be presence or absence of different enendocytic lectins (EELS) and this also the other cell types investigated.
of solasodine glycosides The effect of solamargine on HeLa cells is not inhibited by any of the carbohydrates (Table V) . In addition, solasonine which has a carbohydrate moiety (glycone) Glu(l--4)Ga1(2l)Gal, the di- and monoglycosides [Gal( l-4)Glu,Glu(2- 1)Gal Glu(l-4)Gal, Gal(2- l)Gal, Gal and Glu] and the aglycone solasodine do not inhibit the uptake of tritiated thymidine by HeLa cells (Table I, Fig. 1). This demonstrates that HeLa cells express an EEL for the glycone of solamargine [Gal( l -4)Glu(2l)Gal]. In contrast, the effect of solamargine and DMG (present in BEC) on ovarian cancer cells (Table I) is partially inhibited by lactose (Table V) and lactosyl-albumin (Fig. 1). The lactosyl-albumin inhibitory effect is aproximately 4 times that of lactose at equivalent lactose concentration (Fig. 1). This is because glycoconjugates have an increased affinity for their corresponding lectin relative to unconjugated carbohydrates [32].
214
bed
100
90 80
i
10 0
/0 suhival
60 so 40
UM / L
solamargine/solasodino
Fis. 1. Inhibition of solasodine glycosides (BEC mixture expressed in terms of solamargine) cytotoxicity (uptake of tritiated thymidine) by lactose and lactosyl-albumin. a / ovarian cancer cell line; aI / ovarian cancer cell line in the presence of lactose 1.1 PM/I; a2 / ovarian cancer cell line in the presence of factosylalbumin 1.1 FM/I lactose. Top curve / effect of aglycone solasodine; b / Hela cells; c / lymphoblastoid cells; d / fibroblasts.
100
bed
90 80, 70,
x survival
a
60, so, 40, 30, 20, 10.
s
10 uM
1s
/L
20
2s
* 30
sol8sodine
F&. 2. Effect of high doses of solasodine (aglycone) on the uptake of tritiated thymidine by: a / ovarian cancer cell line; b / HeLa cells; c / lymphoblastoid cells; d / fibroblasts.
215
b
d a
uM/L Fig. 3.
sola
sodine
Effect of high doses of solasodine
lymphocytes.
a / unstimulated;
(aglycone) on the uptake of tritiated thymidine b / PHA; c / Con A; d / PWM.
Similarly, lactose and galactose cause partial inhibition of BEC activity for lymphoblastoid cells as well as ovarian cancer cells (Table V), whereas solasonine and the aglycone are not effective (Table I, Fig. 5). This suggests that ovarian cancer cells and lymphoblastoid express EELS for the glycone of solamargine and also EELs for lactose and galactose (Tables I and V) . The EELS for lactose and galactose may also recognize their respective carbohydrate moieties on the solamargine glycone [lactose = Gal(l-4)GIu]. Similarly, this also applies to fibroblasts for which rhamnose is also inhibitory (Table V). Although rhamnose is not found in mammalian glycoconjugates, it can substitute for fl-galactose under certain conditions [30]. The aglycone solasodine does not inhibit the uptake of tritiated thymidine at equivalent concentrations of solamargine (Fig. 1). However, at higher concentrations there is an inhibitory effect, but this is more apparent with ovarian cancer cells (Fig. 2). This may be explained by
by unstimulated
and stimulated
the changes in membrane permeability of cancer cells [16]. It is possible that solasodine, which is a very hydrophobic molecule, undergoes enhanced protein-binding and thus reduces its bioavailability. The inhibitory effects therefore become apparent at higher concentrations. Similarly, in the case of lymphocytes, the aglycone solasodine does not exert an inhibitory effect at equivalent concentrations of DMG. However, at much higher concentrations, there is a substantial effect on unstimulated lymphocytes, which is significantly reduced in the case of stimulated lymphocytes (Fig. 3). This suggests that membrane permeability to the aglycone is decreased in stimulated lymphocytes. However, this would not account for the differential effect of the glycosides on unstimulated and stimulated lymphocytes (Table II) and inhibition by carbohydrates (Table VI). Both glucose and rhamnose inhibit the action of DMG with Con A stimulated lymphocytes, but not unstimulated lymphocytes. This suggests
216
Table VI. Percentage of tritiated thymidine uptake (cell survival) of unstimulated in the presence of solasodine glycosides and carbohydrates.
US Con A
lymphocytes
DMG Cont.
Percentage
WW
No carbohydrate
Lactose (1.1 /WI)
Galactose (1.1 /WI)
Glucose (1.1 ctM/I)
Rhamnose (1.1 sM/l)
4.81 4.81
72 zt 12 85 EIZ11
69 ZIZ12 75 f 5.5
77 f 8.6 84 ZIE11.8
76 zt 15 109 l 15’
85 f 102 f
n = 7. l0.01 < P > 0.001. l ‘0.02 < P > 0.01. DMG = di- and monoglycosides;
Survival
US = unstimulated;
Ovarian cancer cells in the presence
12 11”
Con A = stimulated.
that unstimulated lymphocytes may express EELS for the carbohydrate moieties of the diglycosides Glu(2- 1)Gal and Glu(l-4)Gal, but not Gal( l -4)Glu (lactose), since lactose was not inhibitory (Table VI). Therefore, the inhibition of DMG by glucose and rhamnose for Con A
Fig. 4.
and Con A-stimulated
of the aglycone
stimulated lymphocytes suggests that Con A stimulation results in the expression of additional EELS for glucose and galactose (Table VI). It is established that Con A stimulation gives rise to a subpopulation of T suppressor cells (TS) that inhibit helper T cells (TH) within the total popuia-
solasodine
96.8 PM/I 3 h
(X 1000).
217
Fig. 5.
Ovarian cancer cells in the presence
of solasodine
glycosides
expressed
in terms of solamargine
6.1 FM/I 3
h (x1000).
tion [17]. The TS cells produce soluble factors that inhibit TH cell functions [Zl, 15,371, but these suppressor factors can be inhibited by IVacetyl-glucosamine or rhamnose by complexing the TH cell receptors [3,X]. Therefore, one of the soluble TS cell factors and/or Con A may be involved in receptor (EEL) induction on TH cells. In addition, since solamargine has a limited inhibitory effect on the uptake of tritiated thymidine by unstimulated lymphocytes (Table II), they may also express an EEL for Gal(l-4)Glu(Z1)Gal. Cytology
The aglycone solasodine did not have any observable effect on the ovarian cancer cells at any of the concentrations investigated (Fig. 4). In contrast, with increasing concentration of the solasodine glycosides (expressed as concentration of solamargine) over 3 h, the cytoplasm of the cancer cells undergo dissolution, the nuclei contract and become dark staining (Fig. 5),
nuclei then enlarge, the chromatin clumps, and finally the nuclei disintegrate (Fig. 6). It therefore appears that the inhibition of thymidine uptake by solamargine is the result of cell lysis, which is dependent on the uptake of solamargine by EELS.
Neoglycoprotein conjugates of cytotoxic drugs have been demonstrated to be suitable for the targeting of cytotoxic drugs via endogenous endocytic lectins (EELS) [l&30-32]. However, these studies have used monosaccharide or disaccharide conjugates for drug targeting of EELS which are also expressed by various normal cells [24,25,35]. In contrast, we have demonstrated the existence of a more complex EEL on an ovarian cancer cell line and HeLa cells for the trisaccharide Gal(l--4)Glu(2l)Gal. The ovarian cancer cells also express EELS for lactose and galactose, as demonstrated by
218
Fig. 6.
Ovarian cancer
cells in the presence
of solasodine
glycosides
expressed
in terms of solamagine
15.3 PM, ‘I 3
h (x 1575)
the limited cytotoxicity of the mono- and diglycosides of solasodine and the partial inhibition of BEC cytotoxicity. Nevertheless, it is the expression of the EEL for the trisaccharide [Gal(l-4)Glu(2l)Gal] of the solasodine glycoside solamargine which appears mainly responsible for the increased cytotoxicity of BEC . This is the first time that an EEL for a trisaccharide has been demonstrated on cancer cells relative to normal cells such that the difference in EEL expression may be exploited for increased specific targeting of cytotoxic glycoconjugates. the natural glycosides of In this respect, solasodine offers a starting point for the investigation of EELS on other types of cancer cells. Also, the lytic action of the solasodine glycosides offers the potential for further investigations for their use in the treatment of cancer.
That lymphocyte EELs can be modulated also offers the potential for therapeutic intervention in lymphatic disorders. This has been considered by Monsigny et al. [29] in the case of murine leukemia, by the use of glycosylated albumin conjugated to methotrexate. In addition, it may be possible to regulate the immune response by non-toxic neoglycoconjugates, such that helper and suppressor activities may be modulated. Ackaowiedgements We wish to thank Mr. T Hurst and Mrs. B Sanderson for their technical assistance. Referencea 1
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