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Differential effects of cyanohydroxybutene and selenium on normal and neoplastic canine mammary cells in vitro
Toxicology Letters, 69 (1993) 97-105 0
97
1993 Elsevier Science Publishers B.V. All rights reserved 0378-4274/93/$06.00
TOXLET 02930
Differential effects of cyanohydroxybutene and selenium on normal and neoplastic canine mammary cells in vitro
M.A. Wallig”, M.J. Kuchanby’ and J.A. Milne?‘*’ Departments of BYeterinary Pathobiology and bFood Science d: Division of Nutritional Sciences, University of Illinois, Urbana, IL (USA)
(Received 10 November 1992) (Accepted 20 January 1993) Key words: 1-Cy~o-Z-hydroxy-3-butene; cell; Glutathione
Selenium; Canine rn~rna~
tumor; Normal canine Mornay
SUMMARY The in vitro responses of canine mammary tumor (CMT- 13) cells and normal canine mammary (NCM) cells to I-cyano-2-hydroxy-3-butene (CHB), a nattily occurring nitrile in cruciterous plants, and &emum (Se) were investigated. CHB at 10 and 20 mM inhibited growth and viability of CMT-13 and NCM cells, respectively. This differential sensitivity was associated with a decreased ability of CMT-13 cells to increase intracellular glutathione (GSH) in comparison to NCM cells. Exposure of both cell types to 3.2 pM Se as sodium selenite alone had no effects, but addition of 3.2 uM Se 24 h after exposure to non-toxic doses of CHB resulted in a substantial decrease in growth of CMT cells, while NCM cells remained unaffected. The synergy noted between CHB and Se in inhibi~g growth and viability of neoplastic mammary cells at levels not toxic to normal mammary cells is promising initial evidence that CHB could have a role in chemoprotection or chemotherapy.
The influence of diet on the development of cancer is currently an active area of investigation. Several epidemiological studies have linked human consumption of Co~respo~e~ce to: Dr. Matthew A. Wallig, Department of Veterinary PathobioIo~, Medicine, 2001 South Lincoln Avenue, Urbana, IL 61801, USA.
College of Veterinary
‘Present address: Department of Nutrition, The Pennsylvania State University, University Park, PA 16802,
USA.
98
cruciferous plants to the decreased incidence of certain tumors [1,2], and some experimental studies in rodents have shown dietary crucifers to prevent certain chemically induced tumors [3]. Some specific compounds from cruciferous plants, mainly glucosinolate breakdown products, have been shown experimentally to possess anticarcinogenic properties [4,_5]. The nitrile, 1-cyano-2-hydroxy-3-butene (CHB), is a naturally occurring glucosinolate breakdown product which forms during the autolysis of certain cruciferous plants. While concentrations of 50 ppm CHB are common in edible crucifers like cauliflower and brussels sprouts, up to 2900 ppm may occur in animal feeds derived from industrial rapeseed and crambe [6]. Studies using the rat as a model have indicated that CHB can induce apoptosis (programmed cell death) in tissues such as pancreas [7]. This ability to induce apoptosis is a characteristic shared by some chemotherapeutic agents such as cisplatin and methotrexate [8]. The pancreatic lesions caused by minimally toxic doses of CHB are completely reversible in the rat, but levels of reduced glutathione (GSH) remain elevated for 4-7 days regardless of the reversibility of the lesions [9,10]. The persistent nature of this elevation of GSH is unique and may indicate potential as an anticarcinogenic or chemoprotective agent, preventing initiation by reactive carcinogens which are detoxified by GSH-mediated conjugation. The trace element, selenium (Se), has been shown in a variety of epidemiological and experimental studies to be a potent anticarcinogen, and the level of dietary Se correlates inversely with cancer mortality and tumor induction by chemical carcinogens [l 1,121. Se is also present at high concentrations in cruciferous plants [ 121.There is accumulating evidence that the cytotoxic effects of Se are due to the generation of toxic GSH-Se intermediates, including selenodiglutathione [ 13,141. Interactions between Se and bioactive glucosinolate breakdown products like CHB have not been investigated, and the contribution of either, or both compounds in the chemoprotection or chemoprevention associated with cruciferous plants remains unknown. MATERIALS
AND METHODS
Cell culture
The canine mammary tumor cell line 13 (CMT-13) was established and characterized by Dr. J. Watrach [14]. Cultures of CMT-13 and primary normal canine mammary (NCM) cells were routinely grown and maintained as previously described [15]. NCM cells were not passed more than three times prior to use in these experiments. Cultures of CMT-13 and NCM cells were allowed to grow for 24 or 48 h, respectively, prior to experimental treatment. This compensated for the longer lag phase encountered with newly plated NCM cultures. Fresh growth medium was added to all cultures 24 h prior to the addition of CHB. Stock solutions of CHB and sodium selenite were dissolved in selenium-free water, filter-sterilized and added in 100 ul aliquots to growth medium. Treatments were generally administered in replicates of four. Cultures of CMT-13 cells were harvested by incubation in 2.5 ml of 0.025% trypsinEDTA for 7 min. NCM cells were far more sensitive to the proteolytic effects of
99
trypsin, and were therefore harvested by incubation in 2.5 ml of 0.012% trypsinEDTA for 2 min. Cell numbers were determined electronically using a Coulter counter, and viabilities were estimated by counting Trypan blue stained suspensions on a hemocytometer. CHB puriJication CHB was isolated from the seed of Abyssinian kale (Crambe abyssinica) [161 and generously supplied by M.E. Daxenbichler and G.F. Spencer at the US Department of Agriculture, National Center for Agricultural Utilization Research, Peoria, IL. The compound was verified as CHB by mass spectrometry and was present exclusively as the S isomer [7]. Glutathione determination Cellular reduced glutathione (GSH) content was assayed by the GSH S-transferase-dependent method of Asaoka and Takahashi [17], using equine GSH S-transferase (Sigma Chemical Co., St. Louis, MO) and with absorbance determined at 540 nm (DU-40 spectrophotometer, Beckman, Fullerton, CA). Estimation of cellular GSH was determined from a standard generated with known concentrations of GSH treated with 5-sulfosalicylic acid and NaOH and expressed as nmol GSH per lo6 viable cells. Experimental design In order to determine whether CHB could differentially affect the growth of NCM cells when compared to CMT-13 cells, both cell types were grown for 24 h in the presence of CHB at concentrations of 0.01, 0.1, 1.0, 2.5, 5.0, 10 and 20 mM. Cell numbers, viability and intracellular GSH were measured at the time of harvest. The influence of combined CHB and selenite on the growth of CMT-13 vs. NCM cells was then assessed by exposing CMT-13 and NCM cells to neither CHB nor Se, to 0.5, 1 mM or 2.5 mM CHB, to 3.2 uM Se (as sodium selenite) or to a combined regimen of 0.5, 1 mM or 2.5 mM CHB + 3.2 uM selenite. The doses of CHB used were selected based on the results of the previous experiment, in which 2.5 mM was the maximal dose at which both CMT-13 and NCM cells were unaffected after 24 h of growth. Likewise, the dose of sodium selenite was chosen to be one at which negative growth of both cell types has not been observed [15]. CMT-13 and NCM cells were seeded at concentrations of 2 and 3 x lo5 cells per 75 cm2 flask, respectively. CHB was added 24 h after seeding (day l), followed by selenite 24 h later (day 2). Cultures were harvested 3, 4 or 5 days after seeding. Cell numbers were measured at the time of harvest. Statistics Data was expressed as mean * SE for four replicates. Unless otherwise stated in the figures, significant differences from control at a particular dose or time-point for a
100
0
NCM
0
cm-13
.
0 .
*
Ol
0
1
I
10
20
Dose
B
CHB
(mM)
1 120 0 0
NCM CMT-13 l
. 60 S 40 -
20 -
0
I 20
I 10
0
Dose
CHB
(mM)
Fig. 1. Influence of CHB on the growth (A) and % viability (B) in CMT-13 and NCM cells. CMT-13 and NCM cultures were seeded at 3.3 and 4.0 x lo5 cells per 25 cm* flask, respectively. Cell numbers and % viability were determined after a 24-h exposure to concentrations of CHB ranging from 0.01 to 20 mM. Control cell numbers were 10.8 x lo2 and 9.8 x 10’ for CMT-13 and NCM cells, respectively. Data represent mean t SE for each dose group. *Indicates a significant difference from control (P < 0.05).
101
particular cell type were detected by one-way ANOVA followed by Fisher’s least significant difference test at the selected doses or time-points. RESULTS
At 0.01 and 0.1 mM CHB neither cell type was adversely affected (Fig. 1A) after 24 h. At 2.5 mM, growth of CMT-13 cells was slightly but not significantly enhanced and NCM cells unaffected. At 5 mM CHB, however, growth of CMT-13 cells was significantly inhibited by approx. 40%. Growth of NCM cells, however, was not significantly affected at this dose. Growth and viability of both cell types was inhibited at 10 and 20 mM CHB, although growth and viability (Fig. 1B) of CMT-13 cells was affected to a greater extent. Baseline intracellular GSH in both cell types was not affected by CHB at doses up to 1.OmM (Fig. 2). GSH was increased above baseline levels, however, at 2.5 and 5.0 mM CHB in NCM and CMT- 13 cells, respectively. NCM cells were capable of markedly increasing baseline GSH con~n~ations in response to CHB, with GSH concentrations steadily rising to >lOOO%above baseline at 20 mM CHB. By contrast, CMT13 cells were unable to increase GSH by more than the initial 250% increase observed at 2.5 mM.
Fig. 2. Influence of CHB on GSH concentrations. GSH was determined after a 24-h exposure to concentrations of CHB ranging from 0.01 to 20 mM. GSH concentrations in control cetls were 35.1 and 13.8 fmol GSHhiable cell for CMT-13 and NCM, respectively. *Indicates a significant difference from control (P < 0.05).
102
q time R
CHB
a
Se
Ed CHB+Se
CMT-I3
NCM
Fig. 3. Intluence of a combination of CHB and selenite on the growth of CMT-I 3 and NCM cells. Cultures were seeded at 2 and 3 x IO5cells per 75 cm2 flask for CMT-13 and NCM cetls, respectively. CHB (1 mM) was added on day 0 followed by selenite (3.2 FM) at 24 h. Data represent mean + SE of measurements on four separate flasks. *Indicates a mean significantly different (P c: 0.05) from the control containing neither CHB nor selenite.
Effects
of CHB -k Se on growth of CMT-IS
and NCM
cells
Preliminary experiments indicated that the two mammary cell types responded differently to a combined regimen of CHB and selenite. Neither 1.OmM CHB nor 3.2 pM Se alone inhibited the growth either mammary cell type (Fig. 3). The addition of 3.2 PM Se after 24 h of exposure to 1.OmM CHB, for example, significantly inhibited the growth of CMT- 13 cells over the next 3 days, with a decrease in ceil growth of 8 1% in comparison to the CHB--Se- controls. In contrast, CHB at 1.OmM followed by 3.2 PM selenite did not affect the growth of NCM cells. The increased sensitivity of CMT-13 cells to combined CHB-Se treatment was also present at 0.5 mM CHB, in which a 42% reduction in growth was observed in the CMT-I 3 cells (data not shown). The growth of NCM cultures was only inhibited at CHB concentrations > 1.O mM
103
CHB, with a 39% reduction in growth when supplemental selenite was added to the culture medium 24 h after 2.5 mM CHB (data not shown). DISCUSSION
In the present experiments, relatively high concentrations of CHB were required in order to observe differences in toxicity between normal and neoplastic mammary cells in short-term cultitres. Nevertheless, a small but significant difference in sensitivity to CHB alone is present between the two cell types, a difference that becomes more apparent over time and with increasing dose. The increased sensitivity of CMT-13 is reflected both as decreased growth and decreased cell viability. A striking differential effect between the two cell types, however, was the inability of CMT-13 cells to increase intracellular GSH concentrations in a steady, dose-dependent fashion when exposed to CHB. The response in the more resistant NCM cells was similar in degree to that observed in the pancreatic acinar cells in vivo after both single and repeated doses of CHB [7,9]. It is not known whether the increase in GSH in both cell types was preceded by a substantial depletion of GSH i~ediately after exposure to CHB, an occurrence observed in liver and pancreas after in vivo exposure to CHB [9]. It has been hypothesized that, due to the chemical structure of CHB, conjugation of CHB to GSH may be the cause of the depletion [9,18]. Depletion of cellular GSH to or below 80%, the point at which cytosolic GSH is almost completely lost [19], has been considered to be sufficient for cell death to occur when a toxic stimulus is present 1201.GSH at time-points earlier than 24 h was not measured in these studies, so it was not possible to determine the degree of depletion in either cell type, whether there was a > 80% depletion of GSH at toxic doses or whether increased synthesis was associated with prior depletion. From the results reported here, it appears that GSH in CMT cells in response to CHB may have attained maximal levels, to the point where GSH became limiting for both the detoxification of CHB and maintenance of growth and viability. The sensitivity of CMT-13 cells to CHB was greatly exacerbated by addition of Se in the form of sodium selenite. A striking synergy was observed between the two compounds, with subtoxic doses of both compounds resulting in a large decrease in the growth of the neoplastic CMT-13 cells when the two compounds were used in combination. There is evidence that the in vitro sensitivity of various ma~ary cell lines to Se corresponds to the ability of the particular cell type or cell line to increase its GSH content [ 151. CMT-13 cells in particular have a decreased ability to increase the activity of the rate-limiting enzyme of GSH synthesis, y-glutamylcysteine synthetase, in response to Se exposure [15], while NCM cells respond in a more typical fashion, with a si~ificant increase in ~-glutamyl~ysteine synthetase. The inability of CMT-I 3 cells in culture to mount a GSH response is associated with enhanced retention of Se and increased sensitivity to the toxic effects of Se [21]. The response of CMT-13 and NCM cells to CHB had a similar pattern to that observed with Se, in which CMT- 13 cells were able to mount only a moderate, 200% maximal GSH in-
104
crease in response to CHB, in contrast to the 1000% response in NCM cells. It is possible that CHB is incompletely metabolized in CMT cells when the additional stress of Se treatment is added, leading to accumulation of not only toxic Se intermediates, but toxic CHB intermediates as well. The lack of sensitivity of NCM cells to combined CHB-Se treatment is initial, positive evidence that CHB and/or a CHB-Se combination might have chemotherapeutic or chemoprotective potential. Furthermore, the marked increase in GSH in the normal cells suggests that this potential may not be limited to pancreas and liver [9]. Induction of tissue GSH has been associated with chemoprotective potential by several investigators [22,23]. Increased GSH has also been effective in reversing the progression of aflatoxin-induced hepatomas [24] and in slowing the growth of rat mammary tumors [25]. In this context, therefore, CHB has potential as a chemopreventive agent by selectively increasing the amount GH in normal tissues available for conjugation to toxicants and/or their reactive intermediates. ACKNOWLEDGEMENT
Supported in part by NIH-NIDDK
grant, DK412 15.
REFERENCES 1 Graham, S. (1983) Results of case-control studies of diet and cancer in Buffalo, New York. Cancer Res. (Suppl.) 43,2409s-2413s. 2 Lee, H.P., Gourley, L., Duffy, S.W., Esteve, J., Lee, J. and Day, N.E. (1989) Colorectal cancer and diet in an Asian population-A case-control study among Singapore Chinese. Int. J. Cancer 43, 1007-1016. 3 Wattenburg, L.W. (1977) Inhibition of carcinogenic effects of polycyclic hydrocarbons by benzyl isothiocyanate and related compounds. J. Natl. Cancer Inst. 58, 395398. 4 Wattenberg, L.W. and Loub, W.D. (1978) Inhibition of polycyclic aromatic hydrocarbon-induced neoplasia by naturally occurring indoles. Cancer Res. 38, 1410-1413. 5 Boyd, J.N., Babish, J.G. and Stoewsand, G.S. (1982) Modification by beet and cabbage diets of aflatoxin B,-induced rat plasma alpha-foetoprotein elevation, hepatic tumorigenesis and mutagenicity of urine. Fd. Chem. Toxicol. 20,47-52. 6 Daxenbichler, M.E., VanEtten, C.H. and Spencer, G.F. (1977) Glucosinolates and derived food products in cruciferous vegetables. Identification of organic nitriles from cabbage. J. Agric. Fd. Chem. 25(l), 121-124. 7 Wallig, M.A., Gould, D.H. and Fettman, M.J. (1988) Selective pancreatotoxicity in the rat induced by the naturally occurring plant nitrile I-cyano-2-hydroxy-3-butene. Fd. Chem. Toxicol. 26(2), 137-147. 8 Barry, M.A., Behnke, C.A. and Eastman, A. (1990) Activation of programmed cell death (apoptosis) by cisplatin, other anticancer drugs, toxins and hyperthermia. Biochem. Pharmacol. 40, 2353-2362. 9 Wallig, M.A. and Jeffery, E.H. (1990) Enhancement of pancreatic and hepatic glutathione levels in rats during cyanohydroxybutene intoxication. Fund. Appl. Toxicol. 14, 144-159. 10 Maher, M., Chemenko, G. and Barrowman, J.A. (1991) The acute pancreatotoxic effects of the plant nitrile I-cyano-2-hydroxy-3-butene. Pancreas 6, 168-174. 11 Schrauzer, G.N., White, D.A. and Schneider, C.J. (1977) Caner mortality correlation studies IV. Association with dietary intakes and blood levels ofcertain trace elements. Notably Se antagonists. Bioinorg. Chem. 7. 35-36.
105
12 Shamberger, R.J. and Willis, C.E. (1971) Selenium distribution and human cancer mortality. CRC Crit. Rev. Clin. Lab. Sci. 2, 21 l-221. 13 Poirier, K.A. and Milner, J.A. (1984) Factors influencing the antitumorigenic properties of selenium in mice. J. Nutr. 113,2147-2154. 14 Fico, M.E., Poirier, K.A., Watrach, A.M., Watrach, M.A. and Milner, J.A. (1986) Differential effects on selenium on normal and neoplastic canine mammary cells. Cancer Res. 46, 33843388. 15 Kuchan, M.J., Fico-Santoro, M. and Milner, J.A. (1990) Consequences of selenite supplementation on the growth and metabolism of cultures of canine mammary cells. J. Nutr. Biochem. 1,478483. 16 Daxenbichler, M.E., VanEtten, C.H. and Wolff, I.A. (1966) (S)- and (R)-1-cyano-2-hydroxy-3-butene from myrosinase hydrolysis of epi-progoitrin and progoitrin. Biochemistry 5(2), 692-697. 17 Asaoka, K. and Takahashi, K. (1981) An enzymatic assay of reduced glutathione Saryltransferase with o-dinitrobenzene as a substrate. J. Biochem. 90, 1237-1242. 18 Talalay, P., De Long, M.J. and Prochaska, H.J. (1988) Identification of a common chemical signal regulating the induction of enzymes that protect against chemical carcinogenesis. Proc. Natl. Acad. Sci. USA 858261-8265. 19 Griffith, O.W. and Meister, A. (1985) Origin and turnover of mitochondrial glutathione. Proc. Natl. Acad. Sci. USA 82,466884672. 20 Mitchell, D.B., Acosta, D. and Bruckner, J.V. (1985) Role of glutathione depletion in the cytotoxicity of acetaminophen in a primary culture system of rat hepatocytes. Toxicology 37, 127-146. 21 Kuchan, M.J. and Milner, J.A. (1991) Influence of supplemental glutathione on selenite-mediated growth inhibition of canine mammary cells. Cancer Lett. 57, 181-186. 22 Brada, Z., Altman, N.H., Hill, M. and Bulba, S. (1982) The effect of methionine on the progression of hepatocellular carcinoma induced by ethionine. Res. Commun. Chem. Pathol. Pharmacol. 38, 157-160. 23 Mgbodile, M.W.K., Holscher, M. and Neal, R.A. (1975) A possible protective role for reduced glutathione in aflatoxin B, toxicity: Effect of pretreatment of rats with phenobarbital and 3-methylcholanthrene on aflatoxin toxicity. Toxicol. Appl. Pharmacol. 34, 128142. 24 Novi, A.M. (1981) Regression of aflatoxin B,-induced hepatocellular carcinomas by reduced glutathione. Science 212, 541-542. 25 Karmali, R.A. (1984) Growth inhibition and prostaglandin metabolism in the R3230AC mammary adenocarcinoma by reduced glutathione. Cancer Biochem. Biophys. 7, 147-154.
97
1993 Elsevier Science Publishers B.V. All rights reserved 0378-4274/93/$06.00
TOXLET 02930
Differential effects of cyanohydroxybutene and selenium on normal and neoplastic canine mammary cells in vitro
M.A. Wallig”, M.J. Kuchanby’ and J.A. Milne?‘*’ Departments of BYeterinary Pathobiology and bFood Science d: Division of Nutritional Sciences, University of Illinois, Urbana, IL (USA)
(Received 10 November 1992) (Accepted 20 January 1993) Key words: 1-Cy~o-Z-hydroxy-3-butene; cell; Glutathione
Selenium; Canine rn~rna~
tumor; Normal canine Mornay
SUMMARY The in vitro responses of canine mammary tumor (CMT- 13) cells and normal canine mammary (NCM) cells to I-cyano-2-hydroxy-3-butene (CHB), a nattily occurring nitrile in cruciterous plants, and &emum (Se) were investigated. CHB at 10 and 20 mM inhibited growth and viability of CMT-13 and NCM cells, respectively. This differential sensitivity was associated with a decreased ability of CMT-13 cells to increase intracellular glutathione (GSH) in comparison to NCM cells. Exposure of both cell types to 3.2 pM Se as sodium selenite alone had no effects, but addition of 3.2 uM Se 24 h after exposure to non-toxic doses of CHB resulted in a substantial decrease in growth of CMT cells, while NCM cells remained unaffected. The synergy noted between CHB and Se in inhibi~g growth and viability of neoplastic mammary cells at levels not toxic to normal mammary cells is promising initial evidence that CHB could have a role in chemoprotection or chemotherapy.
The influence of diet on the development of cancer is currently an active area of investigation. Several epidemiological studies have linked human consumption of Co~respo~e~ce to: Dr. Matthew A. Wallig, Department of Veterinary PathobioIo~, Medicine, 2001 South Lincoln Avenue, Urbana, IL 61801, USA.
College of Veterinary
‘Present address: Department of Nutrition, The Pennsylvania State University, University Park, PA 16802,
USA.
98
cruciferous plants to the decreased incidence of certain tumors [1,2], and some experimental studies in rodents have shown dietary crucifers to prevent certain chemically induced tumors [3]. Some specific compounds from cruciferous plants, mainly glucosinolate breakdown products, have been shown experimentally to possess anticarcinogenic properties [4,_5]. The nitrile, 1-cyano-2-hydroxy-3-butene (CHB), is a naturally occurring glucosinolate breakdown product which forms during the autolysis of certain cruciferous plants. While concentrations of 50 ppm CHB are common in edible crucifers like cauliflower and brussels sprouts, up to 2900 ppm may occur in animal feeds derived from industrial rapeseed and crambe [6]. Studies using the rat as a model have indicated that CHB can induce apoptosis (programmed cell death) in tissues such as pancreas [7]. This ability to induce apoptosis is a characteristic shared by some chemotherapeutic agents such as cisplatin and methotrexate [8]. The pancreatic lesions caused by minimally toxic doses of CHB are completely reversible in the rat, but levels of reduced glutathione (GSH) remain elevated for 4-7 days regardless of the reversibility of the lesions [9,10]. The persistent nature of this elevation of GSH is unique and may indicate potential as an anticarcinogenic or chemoprotective agent, preventing initiation by reactive carcinogens which are detoxified by GSH-mediated conjugation. The trace element, selenium (Se), has been shown in a variety of epidemiological and experimental studies to be a potent anticarcinogen, and the level of dietary Se correlates inversely with cancer mortality and tumor induction by chemical carcinogens [l 1,121. Se is also present at high concentrations in cruciferous plants [ 121.There is accumulating evidence that the cytotoxic effects of Se are due to the generation of toxic GSH-Se intermediates, including selenodiglutathione [ 13,141. Interactions between Se and bioactive glucosinolate breakdown products like CHB have not been investigated, and the contribution of either, or both compounds in the chemoprotection or chemoprevention associated with cruciferous plants remains unknown. MATERIALS
AND METHODS
Cell culture
The canine mammary tumor cell line 13 (CMT-13) was established and characterized by Dr. J. Watrach [14]. Cultures of CMT-13 and primary normal canine mammary (NCM) cells were routinely grown and maintained as previously described [15]. NCM cells were not passed more than three times prior to use in these experiments. Cultures of CMT-13 and NCM cells were allowed to grow for 24 or 48 h, respectively, prior to experimental treatment. This compensated for the longer lag phase encountered with newly plated NCM cultures. Fresh growth medium was added to all cultures 24 h prior to the addition of CHB. Stock solutions of CHB and sodium selenite were dissolved in selenium-free water, filter-sterilized and added in 100 ul aliquots to growth medium. Treatments were generally administered in replicates of four. Cultures of CMT-13 cells were harvested by incubation in 2.5 ml of 0.025% trypsinEDTA for 7 min. NCM cells were far more sensitive to the proteolytic effects of
99
trypsin, and were therefore harvested by incubation in 2.5 ml of 0.012% trypsinEDTA for 2 min. Cell numbers were determined electronically using a Coulter counter, and viabilities were estimated by counting Trypan blue stained suspensions on a hemocytometer. CHB puriJication CHB was isolated from the seed of Abyssinian kale (Crambe abyssinica) [161 and generously supplied by M.E. Daxenbichler and G.F. Spencer at the US Department of Agriculture, National Center for Agricultural Utilization Research, Peoria, IL. The compound was verified as CHB by mass spectrometry and was present exclusively as the S isomer [7]. Glutathione determination Cellular reduced glutathione (GSH) content was assayed by the GSH S-transferase-dependent method of Asaoka and Takahashi [17], using equine GSH S-transferase (Sigma Chemical Co., St. Louis, MO) and with absorbance determined at 540 nm (DU-40 spectrophotometer, Beckman, Fullerton, CA). Estimation of cellular GSH was determined from a standard generated with known concentrations of GSH treated with 5-sulfosalicylic acid and NaOH and expressed as nmol GSH per lo6 viable cells. Experimental design In order to determine whether CHB could differentially affect the growth of NCM cells when compared to CMT-13 cells, both cell types were grown for 24 h in the presence of CHB at concentrations of 0.01, 0.1, 1.0, 2.5, 5.0, 10 and 20 mM. Cell numbers, viability and intracellular GSH were measured at the time of harvest. The influence of combined CHB and selenite on the growth of CMT-13 vs. NCM cells was then assessed by exposing CMT-13 and NCM cells to neither CHB nor Se, to 0.5, 1 mM or 2.5 mM CHB, to 3.2 uM Se (as sodium selenite) or to a combined regimen of 0.5, 1 mM or 2.5 mM CHB + 3.2 uM selenite. The doses of CHB used were selected based on the results of the previous experiment, in which 2.5 mM was the maximal dose at which both CMT-13 and NCM cells were unaffected after 24 h of growth. Likewise, the dose of sodium selenite was chosen to be one at which negative growth of both cell types has not been observed [15]. CMT-13 and NCM cells were seeded at concentrations of 2 and 3 x lo5 cells per 75 cm2 flask, respectively. CHB was added 24 h after seeding (day l), followed by selenite 24 h later (day 2). Cultures were harvested 3, 4 or 5 days after seeding. Cell numbers were measured at the time of harvest. Statistics Data was expressed as mean * SE for four replicates. Unless otherwise stated in the figures, significant differences from control at a particular dose or time-point for a
100
0
NCM
0
cm-13
.
0 .
*
Ol
0
1
I
10
20
Dose
B
CHB
(mM)
1 120 0 0
NCM CMT-13 l
. 60 S 40 -
20 -
0
I 20
I 10
0
Dose
CHB
(mM)
Fig. 1. Influence of CHB on the growth (A) and % viability (B) in CMT-13 and NCM cells. CMT-13 and NCM cultures were seeded at 3.3 and 4.0 x lo5 cells per 25 cm* flask, respectively. Cell numbers and % viability were determined after a 24-h exposure to concentrations of CHB ranging from 0.01 to 20 mM. Control cell numbers were 10.8 x lo2 and 9.8 x 10’ for CMT-13 and NCM cells, respectively. Data represent mean t SE for each dose group. *Indicates a significant difference from control (P < 0.05).
101
particular cell type were detected by one-way ANOVA followed by Fisher’s least significant difference test at the selected doses or time-points. RESULTS
At 0.01 and 0.1 mM CHB neither cell type was adversely affected (Fig. 1A) after 24 h. At 2.5 mM, growth of CMT-13 cells was slightly but not significantly enhanced and NCM cells unaffected. At 5 mM CHB, however, growth of CMT-13 cells was significantly inhibited by approx. 40%. Growth of NCM cells, however, was not significantly affected at this dose. Growth and viability of both cell types was inhibited at 10 and 20 mM CHB, although growth and viability (Fig. 1B) of CMT-13 cells was affected to a greater extent. Baseline intracellular GSH in both cell types was not affected by CHB at doses up to 1.OmM (Fig. 2). GSH was increased above baseline levels, however, at 2.5 and 5.0 mM CHB in NCM and CMT- 13 cells, respectively. NCM cells were capable of markedly increasing baseline GSH con~n~ations in response to CHB, with GSH concentrations steadily rising to >lOOO%above baseline at 20 mM CHB. By contrast, CMT13 cells were unable to increase GSH by more than the initial 250% increase observed at 2.5 mM.
Fig. 2. Influence of CHB on GSH concentrations. GSH was determined after a 24-h exposure to concentrations of CHB ranging from 0.01 to 20 mM. GSH concentrations in control cetls were 35.1 and 13.8 fmol GSHhiable cell for CMT-13 and NCM, respectively. *Indicates a significant difference from control (P < 0.05).
102
q time R
CHB
a
Se
Ed CHB+Se
CMT-I3
NCM
Fig. 3. Intluence of a combination of CHB and selenite on the growth of CMT-I 3 and NCM cells. Cultures were seeded at 2 and 3 x IO5cells per 75 cm2 flask for CMT-13 and NCM cetls, respectively. CHB (1 mM) was added on day 0 followed by selenite (3.2 FM) at 24 h. Data represent mean + SE of measurements on four separate flasks. *Indicates a mean significantly different (P c: 0.05) from the control containing neither CHB nor selenite.
Effects
of CHB -k Se on growth of CMT-IS
and NCM
cells
Preliminary experiments indicated that the two mammary cell types responded differently to a combined regimen of CHB and selenite. Neither 1.OmM CHB nor 3.2 pM Se alone inhibited the growth either mammary cell type (Fig. 3). The addition of 3.2 PM Se after 24 h of exposure to 1.OmM CHB, for example, significantly inhibited the growth of CMT- 13 cells over the next 3 days, with a decrease in ceil growth of 8 1% in comparison to the CHB--Se- controls. In contrast, CHB at 1.OmM followed by 3.2 PM selenite did not affect the growth of NCM cells. The increased sensitivity of CMT-13 cells to combined CHB-Se treatment was also present at 0.5 mM CHB, in which a 42% reduction in growth was observed in the CMT-I 3 cells (data not shown). The growth of NCM cultures was only inhibited at CHB concentrations > 1.O mM
103
CHB, with a 39% reduction in growth when supplemental selenite was added to the culture medium 24 h after 2.5 mM CHB (data not shown). DISCUSSION
In the present experiments, relatively high concentrations of CHB were required in order to observe differences in toxicity between normal and neoplastic mammary cells in short-term cultitres. Nevertheless, a small but significant difference in sensitivity to CHB alone is present between the two cell types, a difference that becomes more apparent over time and with increasing dose. The increased sensitivity of CMT-13 is reflected both as decreased growth and decreased cell viability. A striking differential effect between the two cell types, however, was the inability of CMT-13 cells to increase intracellular GSH concentrations in a steady, dose-dependent fashion when exposed to CHB. The response in the more resistant NCM cells was similar in degree to that observed in the pancreatic acinar cells in vivo after both single and repeated doses of CHB [7,9]. It is not known whether the increase in GSH in both cell types was preceded by a substantial depletion of GSH i~ediately after exposure to CHB, an occurrence observed in liver and pancreas after in vivo exposure to CHB [9]. It has been hypothesized that, due to the chemical structure of CHB, conjugation of CHB to GSH may be the cause of the depletion [9,18]. Depletion of cellular GSH to or below 80%, the point at which cytosolic GSH is almost completely lost [19], has been considered to be sufficient for cell death to occur when a toxic stimulus is present 1201.GSH at time-points earlier than 24 h was not measured in these studies, so it was not possible to determine the degree of depletion in either cell type, whether there was a > 80% depletion of GSH at toxic doses or whether increased synthesis was associated with prior depletion. From the results reported here, it appears that GSH in CMT cells in response to CHB may have attained maximal levels, to the point where GSH became limiting for both the detoxification of CHB and maintenance of growth and viability. The sensitivity of CMT-13 cells to CHB was greatly exacerbated by addition of Se in the form of sodium selenite. A striking synergy was observed between the two compounds, with subtoxic doses of both compounds resulting in a large decrease in the growth of the neoplastic CMT-13 cells when the two compounds were used in combination. There is evidence that the in vitro sensitivity of various ma~ary cell lines to Se corresponds to the ability of the particular cell type or cell line to increase its GSH content [ 151. CMT-13 cells in particular have a decreased ability to increase the activity of the rate-limiting enzyme of GSH synthesis, y-glutamylcysteine synthetase, in response to Se exposure [15], while NCM cells respond in a more typical fashion, with a si~ificant increase in ~-glutamyl~ysteine synthetase. The inability of CMT-I 3 cells in culture to mount a GSH response is associated with enhanced retention of Se and increased sensitivity to the toxic effects of Se [21]. The response of CMT-13 and NCM cells to CHB had a similar pattern to that observed with Se, in which CMT- 13 cells were able to mount only a moderate, 200% maximal GSH in-
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crease in response to CHB, in contrast to the 1000% response in NCM cells. It is possible that CHB is incompletely metabolized in CMT cells when the additional stress of Se treatment is added, leading to accumulation of not only toxic Se intermediates, but toxic CHB intermediates as well. The lack of sensitivity of NCM cells to combined CHB-Se treatment is initial, positive evidence that CHB and/or a CHB-Se combination might have chemotherapeutic or chemoprotective potential. Furthermore, the marked increase in GSH in the normal cells suggests that this potential may not be limited to pancreas and liver [9]. Induction of tissue GSH has been associated with chemoprotective potential by several investigators [22,23]. Increased GSH has also been effective in reversing the progression of aflatoxin-induced hepatomas [24] and in slowing the growth of rat mammary tumors [25]. In this context, therefore, CHB has potential as a chemopreventive agent by selectively increasing the amount GH in normal tissues available for conjugation to toxicants and/or their reactive intermediates. ACKNOWLEDGEMENT
Supported in part by NIH-NIDDK
grant, DK412 15.
REFERENCES 1 Graham, S. (1983) Results of case-control studies of diet and cancer in Buffalo, New York. Cancer Res. (Suppl.) 43,2409s-2413s. 2 Lee, H.P., Gourley, L., Duffy, S.W., Esteve, J., Lee, J. and Day, N.E. (1989) Colorectal cancer and diet in an Asian population-A case-control study among Singapore Chinese. Int. J. Cancer 43, 1007-1016. 3 Wattenburg, L.W. (1977) Inhibition of carcinogenic effects of polycyclic hydrocarbons by benzyl isothiocyanate and related compounds. J. Natl. Cancer Inst. 58, 395398. 4 Wattenberg, L.W. and Loub, W.D. (1978) Inhibition of polycyclic aromatic hydrocarbon-induced neoplasia by naturally occurring indoles. Cancer Res. 38, 1410-1413. 5 Boyd, J.N., Babish, J.G. and Stoewsand, G.S. (1982) Modification by beet and cabbage diets of aflatoxin B,-induced rat plasma alpha-foetoprotein elevation, hepatic tumorigenesis and mutagenicity of urine. Fd. Chem. Toxicol. 20,47-52. 6 Daxenbichler, M.E., VanEtten, C.H. and Spencer, G.F. (1977) Glucosinolates and derived food products in cruciferous vegetables. Identification of organic nitriles from cabbage. J. Agric. Fd. Chem. 25(l), 121-124. 7 Wallig, M.A., Gould, D.H. and Fettman, M.J. (1988) Selective pancreatotoxicity in the rat induced by the naturally occurring plant nitrile I-cyano-2-hydroxy-3-butene. Fd. Chem. Toxicol. 26(2), 137-147. 8 Barry, M.A., Behnke, C.A. and Eastman, A. (1990) Activation of programmed cell death (apoptosis) by cisplatin, other anticancer drugs, toxins and hyperthermia. Biochem. Pharmacol. 40, 2353-2362. 9 Wallig, M.A. and Jeffery, E.H. (1990) Enhancement of pancreatic and hepatic glutathione levels in rats during cyanohydroxybutene intoxication. Fund. Appl. Toxicol. 14, 144-159. 10 Maher, M., Chemenko, G. and Barrowman, J.A. (1991) The acute pancreatotoxic effects of the plant nitrile I-cyano-2-hydroxy-3-butene. Pancreas 6, 168-174. 11 Schrauzer, G.N., White, D.A. and Schneider, C.J. (1977) Caner mortality correlation studies IV. Association with dietary intakes and blood levels ofcertain trace elements. Notably Se antagonists. Bioinorg. Chem. 7. 35-36.
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12 Shamberger, R.J. and Willis, C.E. (1971) Selenium distribution and human cancer mortality. CRC Crit. Rev. Clin. Lab. Sci. 2, 21 l-221. 13 Poirier, K.A. and Milner, J.A. (1984) Factors influencing the antitumorigenic properties of selenium in mice. J. Nutr. 113,2147-2154. 14 Fico, M.E., Poirier, K.A., Watrach, A.M., Watrach, M.A. and Milner, J.A. (1986) Differential effects on selenium on normal and neoplastic canine mammary cells. Cancer Res. 46, 33843388. 15 Kuchan, M.J., Fico-Santoro, M. and Milner, J.A. (1990) Consequences of selenite supplementation on the growth and metabolism of cultures of canine mammary cells. J. Nutr. Biochem. 1,478483. 16 Daxenbichler, M.E., VanEtten, C.H. and Wolff, I.A. (1966) (S)- and (R)-1-cyano-2-hydroxy-3-butene from myrosinase hydrolysis of epi-progoitrin and progoitrin. Biochemistry 5(2), 692-697. 17 Asaoka, K. and Takahashi, K. (1981) An enzymatic assay of reduced glutathione Saryltransferase with o-dinitrobenzene as a substrate. J. Biochem. 90, 1237-1242. 18 Talalay, P., De Long, M.J. and Prochaska, H.J. (1988) Identification of a common chemical signal regulating the induction of enzymes that protect against chemical carcinogenesis. Proc. Natl. Acad. Sci. USA 858261-8265. 19 Griffith, O.W. and Meister, A. (1985) Origin and turnover of mitochondrial glutathione. Proc. Natl. Acad. Sci. USA 82,466884672. 20 Mitchell, D.B., Acosta, D. and Bruckner, J.V. (1985) Role of glutathione depletion in the cytotoxicity of acetaminophen in a primary culture system of rat hepatocytes. Toxicology 37, 127-146. 21 Kuchan, M.J. and Milner, J.A. (1991) Influence of supplemental glutathione on selenite-mediated growth inhibition of canine mammary cells. Cancer Lett. 57, 181-186. 22 Brada, Z., Altman, N.H., Hill, M. and Bulba, S. (1982) The effect of methionine on the progression of hepatocellular carcinoma induced by ethionine. Res. Commun. Chem. Pathol. Pharmacol. 38, 157-160. 23 Mgbodile, M.W.K., Holscher, M. and Neal, R.A. (1975) A possible protective role for reduced glutathione in aflatoxin B, toxicity: Effect of pretreatment of rats with phenobarbital and 3-methylcholanthrene on aflatoxin toxicity. Toxicol. Appl. Pharmacol. 34, 128142. 24 Novi, A.M. (1981) Regression of aflatoxin B,-induced hepatocellular carcinomas by reduced glutathione. Science 212, 541-542. 25 Karmali, R.A. (1984) Growth inhibition and prostaglandin metabolism in the R3230AC mammary adenocarcinoma by reduced glutathione. Cancer Biochem. Biophys. 7, 147-154.