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Ayurvedic (science of life) agents induce differentiation in murine neuroblastoma cells in culture
Neuropharmacology
Vol. 31,
No.
6, pp.
599407,
1992
002%3908/92 $5.00 + 0.00 Copyright 0 1992 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
AYURVEDIC (SCIENCE OF LIFE) AGENTS INDUCE DIFFERENTIATION IN MURINE NEUROBLASTOMA CELLS IN CULTURE K. N.
PRASAD,'
JUDITH EDWARDS-PRASAD,’SUSAN KENTROTI,*C. BRODIE*and ANTONIA VERNADAKIS*
‘Center for Vitamins and Cancer Research, Department of Radiology and *Departments of Psychiatry and Pharmacology, University of Colorado Health Sciences Center, Box C-276, 4200 East Ninth Avenue, Denver, CO 80262, U.S.A. (Accepted 16 December 1991)
Summary-Many Indian Ayurvedic (science of life) agents have been introduced into the U.S.A. as food supplements. Two of them, Maharishi Amrit Kalash-Ambrosia (MAK-A) and Maharishi Amrit Kalash-Nectar (MAK-N) are under investigation. This study shows that an ethanol extract of MAK-A induced morphological (neurite formation) and biochemical (increase of activity of tyrosine hydroxylase by about 15-fold) differentiation in murine neuroblastoma (NBP,) cells in culture, whereas an aqueous extract of MAK-A increased only the activity of tyrosine hydroxylase but to a much lesser extent. The treatment time of 3 days was needed for the expression of maximum differentiation. Ethanol extracts of MAK-A and aqueous extracts of MAK-A increased the intracellular level of adenosine 3’,5’-cyclic monophosphate (CAMP) by about I-fold in 3 days but they did not do so in 15 min. Ethanol extracts of MAK-A also induced neurite formation in neuroblastoma cells grown in serum free medium but the concentration requirement was about a fifth of that needed in serum. The treatment time of 24 hr was sufficient to induce optimal differentiation in neuroblastoma cells grown in serum free medium. The differentiating agents in ethanol-MAK-A were resistant to heat and light and could not be removed by treatment with activated charcoal. Neither ethanol-MAK-N nor aqueous-MAK-N induced differentiation in neuroblastoma cells, suggesting that the differentiating agents were present only in MAK-A. Key words-tyrosine
hydroxylase, neurites, adenosine 3’,5’-cyclic monophosphate.
The beneficial effects of numerous Ayurvedic agents have been described in an ancient Indian medical book, referred to as Ayurveda. The term Ayurveda is derived from two Sanskrit words: Ayur means
life or life span and Veda means knowledge or science and can also be translated as “science of life”. These agents have been used on the Indian subcontinent for the prevention and treatment of human illness for centuries. The biological basis for their effectiveness remains poorly defined. Recently, several Ayurvedic products have been brought into the market in the United States as food supplements; therefore, they could have a significant impact on the health of persons consuming them on a regular basis. Two of these: Maharishi Amrit KalashAmbrosia (MAK-A, also called MAK-5) and Maharishi Amrit Kalash-Nectar (MAK-N, also called MAK-4), have been under investigation. The biochemical ingredients and pharmacological actions of most of the herbs that are present in MAK-A and MAK-N have been described in detail in a recent book (Kapoor, 1990). Preliminary data on animal models showed that the supplementation of diet with 6% of MAK4 reduced the incidence and growth of 7,12-dimethylbenz(a) anthrocene (DMBA)-induced
mammary tumours in rats, without any toxicity (Sharma, Dwivedi, Satter, Gudehithlu, Abou-Issa, Malarkey and Tejwani, 1990). The dietary sup plementation with MAK-A also stimulated cellular immunity in mice when assayed in vitro (Dileepan, Patel, Sharma and Stechschulte, 1991). Neither the effect of MAK-A nor MAK-N on mammalian cells in culture has been studied. This is essential in order to identify a range of cellular responses that can be produced by these Ayurvedic agents. The costly and time-consuming investigations of isolation, purification and eventually synthesis of active components that produce specific biological responses cannot be initiated until these studies are completed. In this study, a well-defined clone, NBPl obtained from murine neuroblastoma cells (Prasad, 1975) has been used in order to identify differentiating and/or growth-inhibiting agents in the extracts of MAK-A and MAK-N. It is now reported that an ethanol extract of MAK-A induced differentiation in neuroblastoma cells in culture; whereas an ethanol extract of another Ayurvedic product, MAK-N did not. An aqueous extract of MAK-A inhibited growth, whereas an aqueous extract of MAK-N did not. 599
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PRASADet al.
METHODS
Cell culture
A mouse neuroblastoma clone, NBP,, was used in this study. This clone has been developed in this laboratory and contains both tyrosine hydroxylase and choline acetyltransferase (Prasad, Mandal, Waymire, Lees, Vernadakis and Weiner, 1973). Cells were grown in F12 medium, having 5% fetal calf serum, streptomycin (100 p g/ml) and penicillin (lOOunits/ml) and were maintained at 37°C in a humidified atmosphere of 5% CO,. The doubling time of neuroblastoma cells, under the above growth conditions was about 18 hr. Ingredients of MAK-A and MAK-N
MAK-A in tablet form and MAK-N in the form of a paste, were supplied by Maharishi AyurVeda Products International (MAPI, Inc., Stoneham, Massachusetts) and were stored at room temperature. The ingredients of MAK-A were media milkweed, heartleaf moonseed, cyperus, Curculigo orchiodes (black musale), Sphaeranthus indicus (East Indian globe thistle), Clitoria terneata (butterfly pea), Glycyrrhiza glabra (licorice), Vanda roxburghil (vanda orchid), Argyreia speciosa (elephant creeper) and Indian wild pepper. The ingredients of MAK-N were Indian gallnut, Indian gooseberry, dried catkins, Hydrocotyle asiateca (Indian pennywort), Cyperus rotundus (nutgrass), Santalum album (white sandalwood), Clitoria terneata, (butterfly pea), A. guiloria (aloe wood), Glycyrrhiza glubra (licorice), Amonum subalatum (cardamon), Curcuma longa (turneric), Cinnamonum zeylanicum (cinnamon), raw sugar and ghee (clarified butter). Neither MAK-A nor MAK-N contained preservatives. Two ingredients, licorice and butterfly pea, were common to MAK-A and MAK-N. Preparation of extracts of MAK
MAK-A was crushed to a powder and the solvents, such as dimethyl sulfoxide, acetone and ethanol, were added to separate aliquots of the powder at a concentration of O.Sg/ml for various intervals of time at room temperature and away from light. Each preparation was vortexed several times and after the incubation, each extract was centrifuged at 1500 g for 10 min, filtered through a 0.45 pm pore-sized filter paper and was tested for its biological activity on the criteria of morphological differentiation and inhibition of growth in neuroblastoma cells in culture. The ethanol extract was found to be most effective with no measurable effect of solvent. An incubation time of 24 hr was found to be optimal and, therefore, this time interval was used for all subsequent extract preparations. The aqueous extract (0.5 g/7.5 ml) of MAK-A was also prepared in a similar manner, except that centrifugation at a higher speed (15,000 g for 30min) was essential in order to facilitate filtration. A greater volume of water was needed,
since the absorption of water by the dry MAK-A powder was rapid. The ethanol and aqueous extracts of MAK-N were prepared in the same manner as those of MAK-A. The paste was weighed and the solvents were added separately. All extracts were stored at 4°C away from light. They were added directly to the culture medium. The extracts were lyophilized and the dried contents were weighed. The concentrations were expressed as pg of extract per ml of culture medium. The dried content of ethanol and water extracts of MAK-A represented 5 and 16% of the original amounts, respectively; whereas the ethanol and water extracts of MAK-N contained 50 and 72% of the original amounts, respectively. The higher levels of extracted materials in the ethanol and water extracts of MAK-N were primarily due to the extraction of lipids in ethanol and sugar in water. The ingredients of MAK-N contained sugar and butter but the ingredients of MAK-A contained neither of these. Assay of morphological dtflerentiation and inhibition of growth
The formation of neurites, 50 pm or more in length, which has been considered as an expression of morphological differentiation, was assayed according to a method described earlier (Prasad et al., 1973). In brief, 50,000 cells were plated in tissue culture dishes (60 mm) and ethanol extract of MAK-A was added 1 day later. The growth medium and ethanolMAK-A were changed after 2 days of treatment and the morphological differentiation was determined at various time intervals, according to the experimental designs. Inhibition of growth is a measure of the sum of the changes in the rate of cell proliferation and the extent of cell death and was determined according to the method described earlier (Prasad et al., 1973). To study the effect of ethanol-MAK-A on the growth of neuroblastoma cells, 50,000 cells were plated in tissue culture dishes (60 mm) and ethanol-MAK-A (50 pg/ml) was added 1 day later. The number of cells per dish was determined by a Coulter Counter every day, as described earlier (Prasad et al., 1973). The growth medium and ethanol-MAK-A were changed after 2 days of treatment. After 3 days of treatment ethanol-MAK-A was removed and the number of cells per dish was determined 1 and 2 days after removal. Assay of tyrosine hydroxylase acetyltransferase (ChAT)
(TH)
and choline
The TH and ChAT are considered to be biochemical markers of differentiation for adrenergic and cholinergic nerve cells, respectively. The activity of tyrosine hydroxylase was measured according to the method of Masserano, Takimoto and Weiner (1983), as modified by Kentroti and Vemadakis (1989). The procedure involves the recovery and assay of r4C01 after decarboxylation with partially purified
Ayurvedic agents and differentiation of NB cells 1001
22
3
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Analysis of data
I
The significant difference between mean values of control and experimental groups was determined, using a one-tailed Student’s t-test at a 0.05 level of significance. RESULTS
Effect of an ethanol extract of MAK-A and MAK-N on morphological dQ$erentiation
\
0
1
2
3
4
5
6
Days After Treatment
Fig. 2. Effect of an ethanol extract of MAK-A on morphological differentiation, as a function of treatment time. Ethanol-MAK-A (50 pg/ml) was added I day after plating. After 3 days of treatment, ethanol-MAK-A was removed and the number of morphologically differentiated cells was determined after 1 and 2 days of removal. Each value represents an average of 9 samples rt:SEM. The sizes of bars for the SEM at some points did not exceed the size of the symbol; therefore, they were not represented. hog aromatic L-amino acid decarboxylase of carboxylabelled dihydroxyphenylalanine, formed from carboxy-labelled tyrosine. The activity of ChAT was measured according to the procedure of Fonnum (1975). The procedure involves conversion of choline to acetylcholine by ChAT, in the presence of [‘4C]acetylcoenzyme A. Assay of level of CAMP
The level of CAMP was determined according to the method of Brown, Albano, Ekins and Sgherzi (1979), using the CAMP-kit from Amersham. After incubation, the cells were washed once with serumfree medium and then 1 ml of 10% trichloracetic acid (TCA) was added, after which the contents were frozen. The lysed cells were removed and centrifuged at 16,500 g for 30 min. The supernatant was washed 5 times with water-saturated ether in order to remove the TCA and an aliquot of this was used for determining the level of CAMP. The pellet was used to determine the level of protein, according to the method of Lowry, Rosebrough, Farr and Randall (1951). Assay of activity of adenylate cyclase
The effect of ethanol-MAK-A on the activity of adenylate cyclase of neuroblastoma cells in vitro was determined by measuring the conversion of alpha[“P]ATP to [32P]cAMP and isolating the products by the method of Salomon, Londos and Rodbell (1974) as modified by Minneman, Hegstrand and Molinoff (1979) and adopted for neuroblastoma cells (Sahu, Edwards-Prasad and Prasad, 1988). The activity of adenylate cyclase was expressed as . pmol/min/mg protein.
This study was performed in the presence and absence of serum. An ethanol extract of MAK-A (ethanol-MAK-A) induced morphological differentiation in neuroblastoma cells, grown in the presence of serum in a dose- and time-dependent manner, whereas an ethanol extract of MAK-N (ethanolMAK-N) did not. The control cultures contained mostly round cells, which were clumped at several locations and the spontaneous differentiation in these cultures was less than 1% (Fig. 1A). Treatment of neuroblastoma cells with ethanol-MAK-A for a period of 3 days caused a marked increase in morphological differentiation, as shown by the formation of long neurites and the enlargement of the size of the soma and nucleus (Fig. 1B). Cell death in treated cultures was not significant until 3 days after treatment. No significant cell death occurred in untreated or solvent-treated cultures, at any time during the experimental period. When ethanol-MAK-A was removed from the culture after 3 days of treatment and examined 1 day later, most of the differentiated neuroblastoma cells exhibited marked degeneration, as seen by the formation of numerous vacuoles and the fragmentation of dendritic processes (Fig. lD),
$
r a
60
1
QI .P 0” 40
/
E
B z s”
20 0 LL0
25
50
75
100
Concentrations of ethanol-MAK-A (@ml)
Fig. 3. Effect of an ethanol extract of MAK-A on morphological differentiation as a function of concentration. Ethanol-MAK-A, at various concentrations, was added I day after plating. The morphological differentiation was determined after 3 days of treatment. The concentration of 75 pg/ml was lethal. Each value represents an average of 9 samples f SEM. The sizes of bars for the SEM at some concentrations, did not exceed the size of the symbol;
therefore, they were not represented.
K. N.
602
PRASADet al.
Table 1. Effect of extracts of Maharishi Amrit Kalash-Ambrosia (MAK-A) and Maharishi Amrit Kalash-Nectar (MAK-N) on the growth of neuroblastoma cells in culture
Treatments
Concentrations @g/ml) needed to inhibit the growth by 50% of control
Ethanol extract of MAK-A Ethanol extract of MAK-A after charcoal treatment Aqueous extract of MAK-A Ethanol extract of MAK-N Aqueous extract of MAK-N
27 & 2* 29 * 2 121 f 3 408 _+4 Inactive
The dose-response curve was first constructed using an average of 3 samples per group. The experiment was repeated 3 times. From each of the 3 curves the concentration needed to reduce the growth by 50% of control was determined. Each value represents a” average of 3 separate determinations. *Standard error of the mean.
leading to a reduction in the number of cells with neurites (Fig. 2). These degenerative changes were not observed during the same period if ethanol-MAK-A was not removed from the culture (Fig. 1C). The differentiated neuroblastoma cells eventually died in culture in about 7 days irrespective of the presence of ethanol-MAK-A. This was consistent with the fact that mature nerve cells, when plated in tissue culture, will degenerate rapidly. The extent of morphological differentiation was concentration-dependent, being optimal at 50pg/ml (Fig. 3) and time-dependent, being maximum after 3 days of treatment (Fig. 2). It should be noted that the dose range between minimum and maximum effects on morphological differentiation and between maximum effect and toxicity was narrow. A concentration of 75 pg/ml was toxic. The treatment of neuroblastoma cells with solvent (0.2ZO.4% ethanol) did not induce morphological differentiation and the treated cells appeared to be like the untreated control cells (data not shown). Boiling ethanol-MAK-A for 10 min or exposing it to light, at room temperature for a period of 24 and
Table 3. Effects of extracts of Maharishi Amrit Kalish-Ambrosia (MAK-A) on the intracellular level of CAMP in neuroblastoma cells in culture Level of CAMP (pmolihrima protein) Treatments
15min
3 davs
Control
13+2* 14* 1 I2_+1
II +2* 15f 2** 23 + I**+
Solvent (0.2% ethanol) Ethanol-MAK-A (25ag/ml) Ethanol-MAK-A (50pg/ml) Aqueous-MAK-A Aqueous-MAK-A
12rt I 18 f 2** 19 f 2**
(85 pg/ml) (85 us/ml)
47 * 49.1 25 + 2*** 42 f 3.”
Cells (50,000 cells for all groups except those which received 50 pg/ml and 170 pg/ml of ethanol-MAK-A, the latter were plated with IO’ cells) plated in tissue culture dishes (60 mm) and a” ethanol extract and an aqueous extract of MAK-A were added separately 24 hr later. The medium and extracts were changed after 2 days of treatment and the level of CAMP was determined after 15min and 3days of treatment. Each value represents an average of 3 samples. Experiments were repeated 3 times and similar changes were observed in the treated groups, in comparison to controls. *Standard error of the mea”. **Significantly different (P < 0.05) from control. ***Significantly different (P < 0.05) from solvent-treated control.
72 hr, did not alter potency in causing morphological differentiation (data not shown). The treatment of ethanol-MAK-A with activated charcoal (20 mg/ml) for 30 min at room temperature did not remove the differentiating agents, even though it adsorbed about 50% of the original materials in the extract. A concentration of 39 pg/ml of charcoal-treated ethanol-MAK-A was needed to induce maximum differentiation (68 + 4%) in neuroblastoma cells.
Table 2. Effects of extracts of Maharishi Amrit Kalish-Ambrosia (MAK-A) on activity of tyrosine hydroxylase in neuroblastoma cells in culture Activity of tyrosine hydroxylase (pmol/hr/mg protein) Treatments Control Solvent (0.2% ethanol) Ethanol-MAK-A (501(g/ml) Ethanol-MAK-A (25 fig/ml) Aqueous-MAK-A (170 pg/ml) Aqueous-MAK-A (85 pgjml)
l5min
3 days
8+ I* 12 * I” 11 f I’* -
8+1 17 + I’* 127 + 9*** 25 + 2’*’ 22 5 4*** 16 i I**
Cells (0.25 x 106) for all groups, except those which received 50 Pg/ml of ethanol-MAK-A and 170 fig/ml of aqueous-MAKA, the latter were plated with 1 x lo6 cells and were plated in tissue culture dishes (IOOmm) and a” ethanol extract and aqueous extract of MAK-A, were added separately 24 hr later. The medium and extracts were changed after 2 days of treatment and the activity of the enzyme was determined after 15 min and 3days of treatment. Each value represents a” average of 4 samples. Experiments were repeated 3 times and similar changes were observed in the treated groups, in comparison to controls. *Standard error of the mea”. l*Significantly different (P < 0.05) from control. ***Significantlydifferent (P < 0.05) from solvent-treated control.
0
1
2
3
4
5
Days After Treatment
Fig. 5. Effect of an ethanol extract of MAK-A on morphological differentiation in serum-free medium, as a function of treatment time. Ethanol-MAK-A was added 1 day after plating and the morphological differentiation was determined at 2 and 3 days after treatment. The treatment time of 3 days was lethal. Ethanol-MAK-A was removed after 1 and 2 days of treatment and the number of morphologically differentiated cells was determined 1, 2 and 3 days later. Each value represents an average of 9 samples f SEM. The sizes of bars for the SEM at some points did not exceed the size of the symbol; therefore, they were not represented. 0-O Control; 0-a treatment with ethanolMAK-A; O---O ethanol-MAK-A not removed; A-A ethanol-MAK-A removed after 1 day of treatment; o-0 ethanol-MAK-A removed 2 days after treatment.
Fig. 1. Photomicrographs of neuroblastoma cells, treated with extracts of MAK-A control cultures, 4 days after plating, contained mostly round cells which grew in clumps and exhibited less than 1% spontaneous morphologically differentiated cells (A); cells treated with an ethanol extract of MAK-A (50 pg/ml), for a period of 3 days, were highly differentiated (B); when ethanol-MAK-A was removed and examined 1 day later, extensive degeneration associated with multiple vacuoles was observed in differentiated neuroblastoma cells (D); these changes were not seen if ethanol-MAK-A was not removed during the same period (C); cells treated with an aqueous extract of MAK-A (85 pg/ml, E) for a period of 3 days did not show morphological differentiation. Magnification: x 450.
603
Fig. 4. Photomicrographs of neuroblastoma (NBP,) cells, treated with an ethanol extract of MAK-A in supplemented serum free medium. Control cultures, 3 days after plating, contained primarily round cells without clumping (A); cells treated with ethanol-MAK-5 (10 pg/ml) for a period of 2 days, showed marked increase in morphological differentiation (B); when ethanol-MAK-A was removed, 2 days after treatment and examined 7 days later, the cells continued to express differentiation (C); when ethanol-MAK-A was removed only 1 day after treatment and examined 8 days later, many cells continued to express morphological differentation (D) with an extensive growth of neurites from a single differentiated neuroblastoma cell (E). Magnification: x 450.
604
Ayurvedic agents and differentiation of NB cells
605
24 hr of treatment and examined 8 days later, the differentiated neuroblastoma cells exhibited an extensive network of neurites (Fig. 4D). Many single large differentiated neuroblastoma cells with numerous outgrowth of neurites were seen (Fig. 4E). Treatment of cells with the same amount of solvent (0.2-0.4% ethanol) as the experimental group did not induce morphological differentiation. Effect of ethanol-MAK-A growth.
-I
0
1
1
!i
b
k
6
Days After Plating
Fig. 6. Effect of an ethanol extract of MAK-A on the growth of neuroblastoma cells, in the presence of serum, as a function of treatment time. Ethanol-MAK-A (50 pg/ml) was added 1 day after treatment and the number of cells per dish was determined every day. After 3 days of treatment the ethanol-MAK was removed and the number of cells per dish was determined 1 and 2 days after removal. Each value represents an average of 9 samples. The sizes of the bars for the SEM did not exceed the size of the symbol; therefore, they were not represented. O----0 control; 0-O treatment with ethanol-MAK-A; A---A ethanol-MAKA removed after 3 days of treatment.
Because of the importance of the type of serum in the modulation of expression of differentiated phenotype in neuroblastoma cells (Prasad, Spuhler, Arnold and Vernadakis, 1979), the effect of ethanolMAK-A was investigated in supplemented serumfree medium, which was developed by Bottenstein and Sato (1979). In serum-free medium, the cells were round but they did not form clumps; some of them had short neurites (Fig. 4A). The spontaneous morphological differentiation in untreated control cells varied from 5 f 2% to 15 f 2%, depending upon the time after plating (Fig. 5). Treatment of cells with ethanol-MAK-A also induced morphological differentiation in serum-free medium (Fig. 4B) in a dose-dependent manner, being optimal at 10 pg/ml; and a time-dependent manner (Fig. 5), being optimal at 2 days after treatment. When ethanol-MAK-A was removed after 2 days of treatment, the number of differentiated cells continued to increase and to elaborate longer and thicker neurites after 7 days of removal of ethanol-MAK-A (Fig. 4C). All cells died if ethanol-MAK-A was not removed after 2 days of treatment in serum-free medium. Treatment of neuroblastoma cells with ethanol-MAK-A in serumfree medium for 24 hr was sufficient to induce morphological differentiation. This was shown by the fact that when ethanol-MAK-A was removed after
and ethanol-MAK-N
on
This study was performed in the presence of fetal calf serum. Ethanol-MAK-A inhibited the growth of neuroblastoma cells in a concentrationand time-dependent manner. A 50% inhibition of growth was achieved by a concentration of 27 + 2 pg/ml of ethanol-MAK-A and 408 f 4 pg/ml of ethanol-MAK-N (Table 1). When ethanolMAK-A was removed after 3 days of treatment, the resistant cells appeared to divide at a rate which is faster than those cells which continued to have ethanol-MAK-A (Fig. 6). Eflect of an aqueous extract of MAK-A and MAK-N on growth and morphological differentiation
This study was performed in the presence of fetal calf serum. An aqueous extract of MAK-A inhibited the growth of neuroblastoma cells in a dose-dependent manner (Table l), without causing morphological differentiation (Fig. 1E). A 50% growth inhibition was achieved by 121 + 3 kg/ml of aqueous-MAK-A. The treatment of cells with aqueous-MAK-N up to concentrations of 1440 pg/ml was ineffective (Table 1). The addition of aqueousMAK-N to cultures produced increased clumping of neuroblastoma cells during growth and many of these clumps were detached after 3 days of treatment, therefore, the effect on growth was determined only 2 days after treatment. Similar amounts of solvent, as that found in aqueous-MAK-N (water containing 46 mg/ml of sugar), when added to the culture for the same period as that for experimental groups, did not alter the growth of neuroblastoma cells. Effect of ethanol- and aqueous-MAK-A on tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT)
The enzymes, TH and ChAT, have been considered as an index of biochemical differentiation and they are rate-limiting enzymes in the synthesis of catecholamines and acetycholine, respectively. The expression of morphological and biochemical differentiation is not always coupled (Prasad, 1975), therefore, the effect of extracts of MAK on TH and ChAT was studied in neuroblastoma cells, grown in the presence of serum. Results showed that ethanol-MAK-A, at concentrations of 50 and 25 pg/ml, increased the activity of TH more than that produced by the solvent (0.2% ethanol) after 3 days of treatment. A slight increase in the activity of the enzyme was seen
K. N. h.UAD et al.
606
as early as 15 min after treatment. Ethanol-MAK-A was more effective than aqueous-MAK-A (Table 2). Neither ethanol-MAK-A nor aqueous-MAK-A significantly affected the activity of ChAT (data not shown). The addition of ethanol-MAK-A directly into the reaction mixture did not alter the activity of TH or ChAT (data not shown). Eflect of ethanol- and aqueous-MAK-A CAMP
on the level of
One of the mechanisms of differentiation in neuroblastoma cells is mediated by CAMP-dependent events (Prasad, 1975; Prashad, Rosenberg, Baskin, Sparkman, Ulrich and Wischmeyer, 1980); therefore, the effect of extracts of MAK-A on the level of CAMP was determined. Cells treated with both ethanol- and aqueous-MAK-A increased the level of CAMP after 3 days of treatment. However, only aqueous-MAR-A increased it slightly, after 15 min of treatment (Table 3). The treatment of cells with solvent (0.2% ethanol) did not alter the level of CAMP. Eject o~ethano~-Md K-A on the activity ofadenyiate cyclase. Since some herbs are known to contain forskolin, the effect of ethanol-MAK-A on differentiation of neuroblastoma cells may be mediated through forskolin. One of the actions of forskolin on neuroblastoma cells involves stimulation of the activity of adenylate cyclase in vitro (Sahu et a/., 1988). Therefore, the effect of ethanol-MAK-A on the activity of adenylate cyclase of neuroblastoma cells in vitro was studied. Results showed that the addition of ethanolMAK-A directly to the reaction mixture of neuroblastoma cells, at concentrations 10 and 50 pgjmi, did not alter the activity of adenylate cyclase, in comparison to solvent-treated control (data not shown). The basal level of activity of adenylate cyclase in neuroblastoma cells was 6 + 3 and forskolin stimulated it to 238 + 18 pmol/min/mg/ protein. DISCUSSION
This study reports that an ethanol extract of MAK-A induced differentiation (morphological and biochemical) associated with inhibition of growth in neurobiastoma cells in culture. This was consistent with the observation that the differentiated neuroblastoma cells are always associated with inhibition of growth, although individual differentiated functions can increase their expression in dividing neuroblastoma cells (Prasad, 1975, 1982). An aqueous extract of MAK-A did not induce differentiation but inhibited oniy growth. This suggests that ethanol-MAK-A contained neuroblastoma celldifferentiating agents, whereas aqueous MAK-A has only growth-inhibiting agents; however, the presence of growth-inhibiting agents, in addition to differentiating agents in ethanol-MAK-A cannot be ruled out.
The major concerns, while working with the herbal extract, include the specificity of the cellular response, the importance of the response and the identity of the active ingredients. The effect of ethanol-MAK-A on the differentiation of neuroblastoma cells is fairly specific, since a similar extract, prepared from another herbal product, MAK-N, did not produce such an effect. The induction of differentiation in neuroblastoma cells in culture by ethanol-MAK-A is an important cellular response, which may have relevance in the study of both cancer biology and neurobiology. The identity of differentiating agents in ethanol-MAK-A has not been investigated in this study; however, several ingredients of most of these herbs and plants have been described in a recent book (Kapoor, 1990). The present study showed that ethanol-MAK-A increased the intracellular level of CAMP after 3 days of treatment but did not do so after 15 min. This suggests that the increase in the level of CAMP was not due to a stimulation of the activity of adenylate cyclase by receptor-specific ligands or by inhibitors of CAMP phosphodiesterase, since a prolonged treatment of neuroblastoma cells with ethanol-MAK-A was needed to increase the level of CAMP, This effect of ethanol-MAK-A was similar to that produced by sodium butyrate, a naturally occurring small fatty acid (Prasad, 1980); however, sodium butyrate did not cause morphological differentiation in this clone (Prasad, 1982). Since ethanol-MAK-A did not stimulate the activity of adenylate cyclase activity of neuroblastoma cells in vitro, it is unlikely that the effect of ethanol-MAK-A on differentiation was mediated by forskolin. Ethanol-MAK-A also induced differentiation in neuroblastoma cells grown in serum-free medium, su~esting that this effect of ethanol-MAK-A was not mediated by serum factors. Another possibility is that ethanol-MAK-A may induce differentiation indirectly by stimulating the release of intracellular factors. The removal of ethanol-MAK-A after the maximum expression of morphological differentiation in neuroblastoma celfs produced different effects, depending upon the presence or the absence of serum. For example, in the presence of serum, the removal of ethanol-MAK-A after 3 days of treatment, caused a rapid degeneration of differentiated neuroblastoma cells within 24 hr. No such degenerative changes were observed if ethanol-MAK-A was not removed during the same period. This suggests that the presence of ethanol-MAK-A in medium containing serum, prevented rapid degeneration of differentiated neuroblastoma cells. In contrast to the observations made in serum, degenerative changes in differentiation neuroblastoma cells was not seen in serumfree medium after the removal of ethanol-MAK-A. On the contrary, when ethanol-MAK-A was removed after 1 or 2 days of treatment, the expression of morphological differentiation in neuroblastoma cells continued to increase in quality and quantity. Even
Ayurvedic agents and dilfferentiation of NB cells
after a week, many differentiated neuroblastoma cells with extensive neurites were observed in this group of cultures. These results suggest that an exposure of neuroblastoma cells to ethanol-MAK-A in serumfree medium for 24 hr or in serum for 3 days, was sufficient to alter the genetic expressions, responsible for maintaining the transformed phenotype of neuroblastoma cells in culture. This alteration then allowed the expression of differentiated phenotype in neuroblastoma cells to be manifested. Previous studies (Prasad, 1975, 1982), have shown that an elevation of the intracellular level of CAMP by prostaglandin E, (PGE,), a stimulator of adenylate cyclase, and by (3-butoxy-4-methoxybenzyl)-2imidazolidinone(R020-1724) an inhibitor of CAMP phosphodiesterase, induced differentiation in neuroblastoma cells similar to that produced by ethanolMAK-A. However, in contrast to MAK-A, these CAMP-stimulating agents are toxic. Further studies are in progress to identify and characterize the differentiating and growth-inhibiting agents that are present in MAK-A. Acknowledgement-This
work
was
suppored
by
the
Lancaster Foundation. REFERENCES
Bottenstein J. E. and Sato G. (1979) Growth of a rat neuroblastoma cell line in serum free supplemented medium. Proc. natn. Acad. Sci. U.S.A. 76: 514-517. Brown B. L., Albano J. D. M., Ekins R. P. and Sgherzi A. M. (1979) A simple and sensitive saturation assay method for the measurement of adenosine 3’5’~cyclic monophosphate. Biochem. J. 121: 561-562. Dileepan K. N., Pate1 V., Sharma H. M. and Stechschulte D. J. (1991) Priming of spleenic lymphocytes after ingestion of an Ayurvedic herbal food supplement; Evidence for an immunomodulatory effect. Biochem. Arch. 6: 267-274.
Fonnum F. J. (1975) A rapid radiochemical method for determination of choline acetyltransferase. J. Neurochem. 24: 407409.
Kapoor L. D. (1990) Handbook of Ayurvedic Medicinal Plants. CRS Press, Boca Raton, Florida.
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Kentroti S. and Vernadakis A. (1989)Growth hormone-releasing hormone influences neuronal expression in the developing chick brain. I. Catecholaminergic neurons. Devl. Brain Res. 49: 275-280.
Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with folin phenol reagent. J. biol. Chem. 193: 265-275. Masserano J. M., Takimoto G. S. and Weiner N. (1983) Tyrosine hydroxylase activity in the brain and adrenal gland of rats following chronic administration of ethanol. Alcohol clin. exp. Res. 7: 294298.
Minneman K. P., Hegstrand L. R. and Molinoff M. A. (1979) The pharmacological specificity of B-l and B-2 adrenergic receptors in rat heart and lung. Molec. Pharmat. 16: 21-23. Prasad K. N. (1975) Differentiation of neuroblastoma cells in culture. Biol. Rev. 50: 129-165. Prasad K. N. (1980) Butyric acid: A small fatty acid with diverse biological functions. Life Sci. 27: 1351-1358. Prasad K. N. (1982) Maturation in neuroblastoma. In: Growth Arrest in Cancer (St011B. A., Ed.), pp. 281-308. Wiley, New York. Prasad K. N., Spuhler K., Arnold E. B. and Vernadakis A. (1979) Modification of response of mouse neuroblastoma cells in culture by serum type. In Vitro 15: 807-812. Prasad K. N., Mandal B., Waymire J. C., Lees G. J., Vernadakis A. and Weiner N. (1973) Basal level of neurotransmitter synthesizing enzymes and effect of cyclic AMP agents on the morphological differentiation of isolated neuroblastoma clones. Nature, New Biol. 241: 117-119. Prashad N., Rosenberg R., Baskin F., Sparkman D., Ulrich M. and Wischmeyer B. (1980) Cyclic adenosine 3’,5’monophosphate-binding protein, a biochemical marker of neuroblastoma differentiation. Cancer Res. 40: 28842889.
Sahu S. K., Edwards-Prasad J. and Prasad K. N. (1988) Vitamin E succinate-induced inhibition of prostaglandinstimulated adenylate cyclase activity in neuroblastoma cells. J. Am. Coil. Nutr. 7, 285-294. Salomon Y., Londos C. and Rodbell M. A. (1974) A highly sensitive adenylate cyclase assay. Analyt. Biochem. 58: 541-548.
Sharma H. M., Dwivedi C., Satter B. C., Gudehithlu K. P., Abou-Issa H. Malarkey W. and Teiwani G. I. (1990) Antineoplatic’properties of Maharishc4, against DBMA: induced mammary tumors in rats. Pharmac. Biochem. Behav. 35, 1-7.
Vol. 31,
No.
6, pp.
599407,
1992
002%3908/92 $5.00 + 0.00 Copyright 0 1992 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
AYURVEDIC (SCIENCE OF LIFE) AGENTS INDUCE DIFFERENTIATION IN MURINE NEUROBLASTOMA CELLS IN CULTURE K. N.
PRASAD,'
JUDITH EDWARDS-PRASAD,’SUSAN KENTROTI,*C. BRODIE*and ANTONIA VERNADAKIS*
‘Center for Vitamins and Cancer Research, Department of Radiology and *Departments of Psychiatry and Pharmacology, University of Colorado Health Sciences Center, Box C-276, 4200 East Ninth Avenue, Denver, CO 80262, U.S.A. (Accepted 16 December 1991)
Summary-Many Indian Ayurvedic (science of life) agents have been introduced into the U.S.A. as food supplements. Two of them, Maharishi Amrit Kalash-Ambrosia (MAK-A) and Maharishi Amrit Kalash-Nectar (MAK-N) are under investigation. This study shows that an ethanol extract of MAK-A induced morphological (neurite formation) and biochemical (increase of activity of tyrosine hydroxylase by about 15-fold) differentiation in murine neuroblastoma (NBP,) cells in culture, whereas an aqueous extract of MAK-A increased only the activity of tyrosine hydroxylase but to a much lesser extent. The treatment time of 3 days was needed for the expression of maximum differentiation. Ethanol extracts of MAK-A and aqueous extracts of MAK-A increased the intracellular level of adenosine 3’,5’-cyclic monophosphate (CAMP) by about I-fold in 3 days but they did not do so in 15 min. Ethanol extracts of MAK-A also induced neurite formation in neuroblastoma cells grown in serum free medium but the concentration requirement was about a fifth of that needed in serum. The treatment time of 24 hr was sufficient to induce optimal differentiation in neuroblastoma cells grown in serum free medium. The differentiating agents in ethanol-MAK-A were resistant to heat and light and could not be removed by treatment with activated charcoal. Neither ethanol-MAK-N nor aqueous-MAK-N induced differentiation in neuroblastoma cells, suggesting that the differentiating agents were present only in MAK-A. Key words-tyrosine
hydroxylase, neurites, adenosine 3’,5’-cyclic monophosphate.
The beneficial effects of numerous Ayurvedic agents have been described in an ancient Indian medical book, referred to as Ayurveda. The term Ayurveda is derived from two Sanskrit words: Ayur means
life or life span and Veda means knowledge or science and can also be translated as “science of life”. These agents have been used on the Indian subcontinent for the prevention and treatment of human illness for centuries. The biological basis for their effectiveness remains poorly defined. Recently, several Ayurvedic products have been brought into the market in the United States as food supplements; therefore, they could have a significant impact on the health of persons consuming them on a regular basis. Two of these: Maharishi Amrit KalashAmbrosia (MAK-A, also called MAK-5) and Maharishi Amrit Kalash-Nectar (MAK-N, also called MAK-4), have been under investigation. The biochemical ingredients and pharmacological actions of most of the herbs that are present in MAK-A and MAK-N have been described in detail in a recent book (Kapoor, 1990). Preliminary data on animal models showed that the supplementation of diet with 6% of MAK4 reduced the incidence and growth of 7,12-dimethylbenz(a) anthrocene (DMBA)-induced
mammary tumours in rats, without any toxicity (Sharma, Dwivedi, Satter, Gudehithlu, Abou-Issa, Malarkey and Tejwani, 1990). The dietary sup plementation with MAK-A also stimulated cellular immunity in mice when assayed in vitro (Dileepan, Patel, Sharma and Stechschulte, 1991). Neither the effect of MAK-A nor MAK-N on mammalian cells in culture has been studied. This is essential in order to identify a range of cellular responses that can be produced by these Ayurvedic agents. The costly and time-consuming investigations of isolation, purification and eventually synthesis of active components that produce specific biological responses cannot be initiated until these studies are completed. In this study, a well-defined clone, NBPl obtained from murine neuroblastoma cells (Prasad, 1975) has been used in order to identify differentiating and/or growth-inhibiting agents in the extracts of MAK-A and MAK-N. It is now reported that an ethanol extract of MAK-A induced differentiation in neuroblastoma cells in culture; whereas an ethanol extract of another Ayurvedic product, MAK-N did not. An aqueous extract of MAK-A inhibited growth, whereas an aqueous extract of MAK-N did not. 599
600
K. N.
PRASADet al.
METHODS
Cell culture
A mouse neuroblastoma clone, NBP,, was used in this study. This clone has been developed in this laboratory and contains both tyrosine hydroxylase and choline acetyltransferase (Prasad, Mandal, Waymire, Lees, Vernadakis and Weiner, 1973). Cells were grown in F12 medium, having 5% fetal calf serum, streptomycin (100 p g/ml) and penicillin (lOOunits/ml) and were maintained at 37°C in a humidified atmosphere of 5% CO,. The doubling time of neuroblastoma cells, under the above growth conditions was about 18 hr. Ingredients of MAK-A and MAK-N
MAK-A in tablet form and MAK-N in the form of a paste, were supplied by Maharishi AyurVeda Products International (MAPI, Inc., Stoneham, Massachusetts) and were stored at room temperature. The ingredients of MAK-A were media milkweed, heartleaf moonseed, cyperus, Curculigo orchiodes (black musale), Sphaeranthus indicus (East Indian globe thistle), Clitoria terneata (butterfly pea), Glycyrrhiza glabra (licorice), Vanda roxburghil (vanda orchid), Argyreia speciosa (elephant creeper) and Indian wild pepper. The ingredients of MAK-N were Indian gallnut, Indian gooseberry, dried catkins, Hydrocotyle asiateca (Indian pennywort), Cyperus rotundus (nutgrass), Santalum album (white sandalwood), Clitoria terneata, (butterfly pea), A. guiloria (aloe wood), Glycyrrhiza glubra (licorice), Amonum subalatum (cardamon), Curcuma longa (turneric), Cinnamonum zeylanicum (cinnamon), raw sugar and ghee (clarified butter). Neither MAK-A nor MAK-N contained preservatives. Two ingredients, licorice and butterfly pea, were common to MAK-A and MAK-N. Preparation of extracts of MAK
MAK-A was crushed to a powder and the solvents, such as dimethyl sulfoxide, acetone and ethanol, were added to separate aliquots of the powder at a concentration of O.Sg/ml for various intervals of time at room temperature and away from light. Each preparation was vortexed several times and after the incubation, each extract was centrifuged at 1500 g for 10 min, filtered through a 0.45 pm pore-sized filter paper and was tested for its biological activity on the criteria of morphological differentiation and inhibition of growth in neuroblastoma cells in culture. The ethanol extract was found to be most effective with no measurable effect of solvent. An incubation time of 24 hr was found to be optimal and, therefore, this time interval was used for all subsequent extract preparations. The aqueous extract (0.5 g/7.5 ml) of MAK-A was also prepared in a similar manner, except that centrifugation at a higher speed (15,000 g for 30min) was essential in order to facilitate filtration. A greater volume of water was needed,
since the absorption of water by the dry MAK-A powder was rapid. The ethanol and aqueous extracts of MAK-N were prepared in the same manner as those of MAK-A. The paste was weighed and the solvents were added separately. All extracts were stored at 4°C away from light. They were added directly to the culture medium. The extracts were lyophilized and the dried contents were weighed. The concentrations were expressed as pg of extract per ml of culture medium. The dried content of ethanol and water extracts of MAK-A represented 5 and 16% of the original amounts, respectively; whereas the ethanol and water extracts of MAK-N contained 50 and 72% of the original amounts, respectively. The higher levels of extracted materials in the ethanol and water extracts of MAK-N were primarily due to the extraction of lipids in ethanol and sugar in water. The ingredients of MAK-N contained sugar and butter but the ingredients of MAK-A contained neither of these. Assay of morphological dtflerentiation and inhibition of growth
The formation of neurites, 50 pm or more in length, which has been considered as an expression of morphological differentiation, was assayed according to a method described earlier (Prasad et al., 1973). In brief, 50,000 cells were plated in tissue culture dishes (60 mm) and ethanol extract of MAK-A was added 1 day later. The growth medium and ethanolMAK-A were changed after 2 days of treatment and the morphological differentiation was determined at various time intervals, according to the experimental designs. Inhibition of growth is a measure of the sum of the changes in the rate of cell proliferation and the extent of cell death and was determined according to the method described earlier (Prasad et al., 1973). To study the effect of ethanol-MAK-A on the growth of neuroblastoma cells, 50,000 cells were plated in tissue culture dishes (60 mm) and ethanol-MAK-A (50 pg/ml) was added 1 day later. The number of cells per dish was determined by a Coulter Counter every day, as described earlier (Prasad et al., 1973). The growth medium and ethanol-MAK-A were changed after 2 days of treatment. After 3 days of treatment ethanol-MAK-A was removed and the number of cells per dish was determined 1 and 2 days after removal. Assay of tyrosine hydroxylase acetyltransferase (ChAT)
(TH)
and choline
The TH and ChAT are considered to be biochemical markers of differentiation for adrenergic and cholinergic nerve cells, respectively. The activity of tyrosine hydroxylase was measured according to the method of Masserano, Takimoto and Weiner (1983), as modified by Kentroti and Vemadakis (1989). The procedure involves the recovery and assay of r4C01 after decarboxylation with partially purified
Ayurvedic agents and differentiation of NB cells 1001
22
3
601
Analysis of data
I
The significant difference between mean values of control and experimental groups was determined, using a one-tailed Student’s t-test at a 0.05 level of significance. RESULTS
Effect of an ethanol extract of MAK-A and MAK-N on morphological dQ$erentiation
\
0
1
2
3
4
5
6
Days After Treatment
Fig. 2. Effect of an ethanol extract of MAK-A on morphological differentiation, as a function of treatment time. Ethanol-MAK-A (50 pg/ml) was added I day after plating. After 3 days of treatment, ethanol-MAK-A was removed and the number of morphologically differentiated cells was determined after 1 and 2 days of removal. Each value represents an average of 9 samples rt:SEM. The sizes of bars for the SEM at some points did not exceed the size of the symbol; therefore, they were not represented. hog aromatic L-amino acid decarboxylase of carboxylabelled dihydroxyphenylalanine, formed from carboxy-labelled tyrosine. The activity of ChAT was measured according to the procedure of Fonnum (1975). The procedure involves conversion of choline to acetylcholine by ChAT, in the presence of [‘4C]acetylcoenzyme A. Assay of level of CAMP
The level of CAMP was determined according to the method of Brown, Albano, Ekins and Sgherzi (1979), using the CAMP-kit from Amersham. After incubation, the cells were washed once with serumfree medium and then 1 ml of 10% trichloracetic acid (TCA) was added, after which the contents were frozen. The lysed cells were removed and centrifuged at 16,500 g for 30 min. The supernatant was washed 5 times with water-saturated ether in order to remove the TCA and an aliquot of this was used for determining the level of CAMP. The pellet was used to determine the level of protein, according to the method of Lowry, Rosebrough, Farr and Randall (1951). Assay of activity of adenylate cyclase
The effect of ethanol-MAK-A on the activity of adenylate cyclase of neuroblastoma cells in vitro was determined by measuring the conversion of alpha[“P]ATP to [32P]cAMP and isolating the products by the method of Salomon, Londos and Rodbell (1974) as modified by Minneman, Hegstrand and Molinoff (1979) and adopted for neuroblastoma cells (Sahu, Edwards-Prasad and Prasad, 1988). The activity of adenylate cyclase was expressed as . pmol/min/mg protein.
This study was performed in the presence and absence of serum. An ethanol extract of MAK-A (ethanol-MAK-A) induced morphological differentiation in neuroblastoma cells, grown in the presence of serum in a dose- and time-dependent manner, whereas an ethanol extract of MAK-N (ethanolMAK-N) did not. The control cultures contained mostly round cells, which were clumped at several locations and the spontaneous differentiation in these cultures was less than 1% (Fig. 1A). Treatment of neuroblastoma cells with ethanol-MAK-A for a period of 3 days caused a marked increase in morphological differentiation, as shown by the formation of long neurites and the enlargement of the size of the soma and nucleus (Fig. 1B). Cell death in treated cultures was not significant until 3 days after treatment. No significant cell death occurred in untreated or solvent-treated cultures, at any time during the experimental period. When ethanol-MAK-A was removed from the culture after 3 days of treatment and examined 1 day later, most of the differentiated neuroblastoma cells exhibited marked degeneration, as seen by the formation of numerous vacuoles and the fragmentation of dendritic processes (Fig. lD),
$
r a
60
1
QI .P 0” 40
/
E
B z s”
20 0 LL0
25
50
75
100
Concentrations of ethanol-MAK-A (@ml)
Fig. 3. Effect of an ethanol extract of MAK-A on morphological differentiation as a function of concentration. Ethanol-MAK-A, at various concentrations, was added I day after plating. The morphological differentiation was determined after 3 days of treatment. The concentration of 75 pg/ml was lethal. Each value represents an average of 9 samples f SEM. The sizes of bars for the SEM at some concentrations, did not exceed the size of the symbol;
therefore, they were not represented.
K. N.
602
PRASADet al.
Table 1. Effect of extracts of Maharishi Amrit Kalash-Ambrosia (MAK-A) and Maharishi Amrit Kalash-Nectar (MAK-N) on the growth of neuroblastoma cells in culture
Treatments
Concentrations @g/ml) needed to inhibit the growth by 50% of control
Ethanol extract of MAK-A Ethanol extract of MAK-A after charcoal treatment Aqueous extract of MAK-A Ethanol extract of MAK-N Aqueous extract of MAK-N
27 & 2* 29 * 2 121 f 3 408 _+4 Inactive
The dose-response curve was first constructed using an average of 3 samples per group. The experiment was repeated 3 times. From each of the 3 curves the concentration needed to reduce the growth by 50% of control was determined. Each value represents a” average of 3 separate determinations. *Standard error of the mean.
leading to a reduction in the number of cells with neurites (Fig. 2). These degenerative changes were not observed during the same period if ethanol-MAK-A was not removed from the culture (Fig. 1C). The differentiated neuroblastoma cells eventually died in culture in about 7 days irrespective of the presence of ethanol-MAK-A. This was consistent with the fact that mature nerve cells, when plated in tissue culture, will degenerate rapidly. The extent of morphological differentiation was concentration-dependent, being optimal at 50pg/ml (Fig. 3) and time-dependent, being maximum after 3 days of treatment (Fig. 2). It should be noted that the dose range between minimum and maximum effects on morphological differentiation and between maximum effect and toxicity was narrow. A concentration of 75 pg/ml was toxic. The treatment of neuroblastoma cells with solvent (0.2ZO.4% ethanol) did not induce morphological differentiation and the treated cells appeared to be like the untreated control cells (data not shown). Boiling ethanol-MAK-A for 10 min or exposing it to light, at room temperature for a period of 24 and
Table 3. Effects of extracts of Maharishi Amrit Kalish-Ambrosia (MAK-A) on the intracellular level of CAMP in neuroblastoma cells in culture Level of CAMP (pmolihrima protein) Treatments
15min
3 davs
Control
13+2* 14* 1 I2_+1
II +2* 15f 2** 23 + I**+
Solvent (0.2% ethanol) Ethanol-MAK-A (25ag/ml) Ethanol-MAK-A (50pg/ml) Aqueous-MAK-A Aqueous-MAK-A
12rt I 18 f 2** 19 f 2**
(85 pg/ml) (85 us/ml)
47 * 49.1 25 + 2*** 42 f 3.”
Cells (50,000 cells for all groups except those which received 50 pg/ml and 170 pg/ml of ethanol-MAK-A, the latter were plated with IO’ cells) plated in tissue culture dishes (60 mm) and a” ethanol extract and an aqueous extract of MAK-A were added separately 24 hr later. The medium and extracts were changed after 2 days of treatment and the level of CAMP was determined after 15min and 3days of treatment. Each value represents an average of 3 samples. Experiments were repeated 3 times and similar changes were observed in the treated groups, in comparison to controls. *Standard error of the mea”. **Significantly different (P < 0.05) from control. ***Significantly different (P < 0.05) from solvent-treated control.
72 hr, did not alter potency in causing morphological differentiation (data not shown). The treatment of ethanol-MAK-A with activated charcoal (20 mg/ml) for 30 min at room temperature did not remove the differentiating agents, even though it adsorbed about 50% of the original materials in the extract. A concentration of 39 pg/ml of charcoal-treated ethanol-MAK-A was needed to induce maximum differentiation (68 + 4%) in neuroblastoma cells.
Table 2. Effects of extracts of Maharishi Amrit Kalish-Ambrosia (MAK-A) on activity of tyrosine hydroxylase in neuroblastoma cells in culture Activity of tyrosine hydroxylase (pmol/hr/mg protein) Treatments Control Solvent (0.2% ethanol) Ethanol-MAK-A (501(g/ml) Ethanol-MAK-A (25 fig/ml) Aqueous-MAK-A (170 pg/ml) Aqueous-MAK-A (85 pgjml)
l5min
3 days
8+ I* 12 * I” 11 f I’* -
8+1 17 + I’* 127 + 9*** 25 + 2’*’ 22 5 4*** 16 i I**
Cells (0.25 x 106) for all groups, except those which received 50 Pg/ml of ethanol-MAK-A and 170 fig/ml of aqueous-MAKA, the latter were plated with 1 x lo6 cells and were plated in tissue culture dishes (IOOmm) and a” ethanol extract and aqueous extract of MAK-A, were added separately 24 hr later. The medium and extracts were changed after 2 days of treatment and the activity of the enzyme was determined after 15 min and 3days of treatment. Each value represents a” average of 4 samples. Experiments were repeated 3 times and similar changes were observed in the treated groups, in comparison to controls. *Standard error of the mea”. l*Significantly different (P < 0.05) from control. ***Significantlydifferent (P < 0.05) from solvent-treated control.
0
1
2
3
4
5
Days After Treatment
Fig. 5. Effect of an ethanol extract of MAK-A on morphological differentiation in serum-free medium, as a function of treatment time. Ethanol-MAK-A was added 1 day after plating and the morphological differentiation was determined at 2 and 3 days after treatment. The treatment time of 3 days was lethal. Ethanol-MAK-A was removed after 1 and 2 days of treatment and the number of morphologically differentiated cells was determined 1, 2 and 3 days later. Each value represents an average of 9 samples f SEM. The sizes of bars for the SEM at some points did not exceed the size of the symbol; therefore, they were not represented. 0-O Control; 0-a treatment with ethanolMAK-A; O---O ethanol-MAK-A not removed; A-A ethanol-MAK-A removed after 1 day of treatment; o-0 ethanol-MAK-A removed 2 days after treatment.
Fig. 1. Photomicrographs of neuroblastoma cells, treated with extracts of MAK-A control cultures, 4 days after plating, contained mostly round cells which grew in clumps and exhibited less than 1% spontaneous morphologically differentiated cells (A); cells treated with an ethanol extract of MAK-A (50 pg/ml), for a period of 3 days, were highly differentiated (B); when ethanol-MAK-A was removed and examined 1 day later, extensive degeneration associated with multiple vacuoles was observed in differentiated neuroblastoma cells (D); these changes were not seen if ethanol-MAK-A was not removed during the same period (C); cells treated with an aqueous extract of MAK-A (85 pg/ml, E) for a period of 3 days did not show morphological differentiation. Magnification: x 450.
603
Fig. 4. Photomicrographs of neuroblastoma (NBP,) cells, treated with an ethanol extract of MAK-A in supplemented serum free medium. Control cultures, 3 days after plating, contained primarily round cells without clumping (A); cells treated with ethanol-MAK-5 (10 pg/ml) for a period of 2 days, showed marked increase in morphological differentiation (B); when ethanol-MAK-A was removed, 2 days after treatment and examined 7 days later, the cells continued to express differentiation (C); when ethanol-MAK-A was removed only 1 day after treatment and examined 8 days later, many cells continued to express morphological differentation (D) with an extensive growth of neurites from a single differentiated neuroblastoma cell (E). Magnification: x 450.
604
Ayurvedic agents and differentiation of NB cells
605
24 hr of treatment and examined 8 days later, the differentiated neuroblastoma cells exhibited an extensive network of neurites (Fig. 4D). Many single large differentiated neuroblastoma cells with numerous outgrowth of neurites were seen (Fig. 4E). Treatment of cells with the same amount of solvent (0.2-0.4% ethanol) as the experimental group did not induce morphological differentiation. Effect of ethanol-MAK-A growth.
-I
0
1
1
!i
b
k
6
Days After Plating
Fig. 6. Effect of an ethanol extract of MAK-A on the growth of neuroblastoma cells, in the presence of serum, as a function of treatment time. Ethanol-MAK-A (50 pg/ml) was added 1 day after treatment and the number of cells per dish was determined every day. After 3 days of treatment the ethanol-MAK was removed and the number of cells per dish was determined 1 and 2 days after removal. Each value represents an average of 9 samples. The sizes of the bars for the SEM did not exceed the size of the symbol; therefore, they were not represented. O----0 control; 0-O treatment with ethanol-MAK-A; A---A ethanol-MAKA removed after 3 days of treatment.
Because of the importance of the type of serum in the modulation of expression of differentiated phenotype in neuroblastoma cells (Prasad, Spuhler, Arnold and Vernadakis, 1979), the effect of ethanolMAK-A was investigated in supplemented serumfree medium, which was developed by Bottenstein and Sato (1979). In serum-free medium, the cells were round but they did not form clumps; some of them had short neurites (Fig. 4A). The spontaneous morphological differentiation in untreated control cells varied from 5 f 2% to 15 f 2%, depending upon the time after plating (Fig. 5). Treatment of cells with ethanol-MAK-A also induced morphological differentiation in serum-free medium (Fig. 4B) in a dose-dependent manner, being optimal at 10 pg/ml; and a time-dependent manner (Fig. 5), being optimal at 2 days after treatment. When ethanol-MAK-A was removed after 2 days of treatment, the number of differentiated cells continued to increase and to elaborate longer and thicker neurites after 7 days of removal of ethanol-MAK-A (Fig. 4C). All cells died if ethanol-MAK-A was not removed after 2 days of treatment in serum-free medium. Treatment of neuroblastoma cells with ethanol-MAK-A in serumfree medium for 24 hr was sufficient to induce morphological differentiation. This was shown by the fact that when ethanol-MAK-A was removed after
and ethanol-MAK-N
on
This study was performed in the presence of fetal calf serum. Ethanol-MAK-A inhibited the growth of neuroblastoma cells in a concentrationand time-dependent manner. A 50% inhibition of growth was achieved by a concentration of 27 + 2 pg/ml of ethanol-MAK-A and 408 f 4 pg/ml of ethanol-MAK-N (Table 1). When ethanolMAK-A was removed after 3 days of treatment, the resistant cells appeared to divide at a rate which is faster than those cells which continued to have ethanol-MAK-A (Fig. 6). Eflect of an aqueous extract of MAK-A and MAK-N on growth and morphological differentiation
This study was performed in the presence of fetal calf serum. An aqueous extract of MAK-A inhibited the growth of neuroblastoma cells in a dose-dependent manner (Table l), without causing morphological differentiation (Fig. 1E). A 50% growth inhibition was achieved by 121 + 3 kg/ml of aqueous-MAK-A. The treatment of cells with aqueous-MAK-N up to concentrations of 1440 pg/ml was ineffective (Table 1). The addition of aqueousMAK-N to cultures produced increased clumping of neuroblastoma cells during growth and many of these clumps were detached after 3 days of treatment, therefore, the effect on growth was determined only 2 days after treatment. Similar amounts of solvent, as that found in aqueous-MAK-N (water containing 46 mg/ml of sugar), when added to the culture for the same period as that for experimental groups, did not alter the growth of neuroblastoma cells. Effect of ethanol- and aqueous-MAK-A on tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT)
The enzymes, TH and ChAT, have been considered as an index of biochemical differentiation and they are rate-limiting enzymes in the synthesis of catecholamines and acetycholine, respectively. The expression of morphological and biochemical differentiation is not always coupled (Prasad, 1975), therefore, the effect of extracts of MAK on TH and ChAT was studied in neuroblastoma cells, grown in the presence of serum. Results showed that ethanol-MAK-A, at concentrations of 50 and 25 pg/ml, increased the activity of TH more than that produced by the solvent (0.2% ethanol) after 3 days of treatment. A slight increase in the activity of the enzyme was seen
K. N. h.UAD et al.
606
as early as 15 min after treatment. Ethanol-MAK-A was more effective than aqueous-MAK-A (Table 2). Neither ethanol-MAK-A nor aqueous-MAK-A significantly affected the activity of ChAT (data not shown). The addition of ethanol-MAK-A directly into the reaction mixture did not alter the activity of TH or ChAT (data not shown). Eflect of ethanol- and aqueous-MAK-A CAMP
on the level of
One of the mechanisms of differentiation in neuroblastoma cells is mediated by CAMP-dependent events (Prasad, 1975; Prashad, Rosenberg, Baskin, Sparkman, Ulrich and Wischmeyer, 1980); therefore, the effect of extracts of MAK-A on the level of CAMP was determined. Cells treated with both ethanol- and aqueous-MAK-A increased the level of CAMP after 3 days of treatment. However, only aqueous-MAR-A increased it slightly, after 15 min of treatment (Table 3). The treatment of cells with solvent (0.2% ethanol) did not alter the level of CAMP. Eject o~ethano~-Md K-A on the activity ofadenyiate cyclase. Since some herbs are known to contain forskolin, the effect of ethanol-MAK-A on differentiation of neuroblastoma cells may be mediated through forskolin. One of the actions of forskolin on neuroblastoma cells involves stimulation of the activity of adenylate cyclase in vitro (Sahu et a/., 1988). Therefore, the effect of ethanol-MAK-A on the activity of adenylate cyclase of neuroblastoma cells in vitro was studied. Results showed that the addition of ethanolMAK-A directly to the reaction mixture of neuroblastoma cells, at concentrations 10 and 50 pgjmi, did not alter the activity of adenylate cyclase, in comparison to solvent-treated control (data not shown). The basal level of activity of adenylate cyclase in neuroblastoma cells was 6 + 3 and forskolin stimulated it to 238 + 18 pmol/min/mg/ protein. DISCUSSION
This study reports that an ethanol extract of MAK-A induced differentiation (morphological and biochemical) associated with inhibition of growth in neurobiastoma cells in culture. This was consistent with the observation that the differentiated neuroblastoma cells are always associated with inhibition of growth, although individual differentiated functions can increase their expression in dividing neuroblastoma cells (Prasad, 1975, 1982). An aqueous extract of MAK-A did not induce differentiation but inhibited oniy growth. This suggests that ethanol-MAK-A contained neuroblastoma celldifferentiating agents, whereas aqueous MAK-A has only growth-inhibiting agents; however, the presence of growth-inhibiting agents, in addition to differentiating agents in ethanol-MAK-A cannot be ruled out.
The major concerns, while working with the herbal extract, include the specificity of the cellular response, the importance of the response and the identity of the active ingredients. The effect of ethanol-MAK-A on the differentiation of neuroblastoma cells is fairly specific, since a similar extract, prepared from another herbal product, MAK-N, did not produce such an effect. The induction of differentiation in neuroblastoma cells in culture by ethanol-MAK-A is an important cellular response, which may have relevance in the study of both cancer biology and neurobiology. The identity of differentiating agents in ethanol-MAK-A has not been investigated in this study; however, several ingredients of most of these herbs and plants have been described in a recent book (Kapoor, 1990). The present study showed that ethanol-MAK-A increased the intracellular level of CAMP after 3 days of treatment but did not do so after 15 min. This suggests that the increase in the level of CAMP was not due to a stimulation of the activity of adenylate cyclase by receptor-specific ligands or by inhibitors of CAMP phosphodiesterase, since a prolonged treatment of neuroblastoma cells with ethanol-MAK-A was needed to increase the level of CAMP, This effect of ethanol-MAK-A was similar to that produced by sodium butyrate, a naturally occurring small fatty acid (Prasad, 1980); however, sodium butyrate did not cause morphological differentiation in this clone (Prasad, 1982). Since ethanol-MAK-A did not stimulate the activity of adenylate cyclase activity of neuroblastoma cells in vitro, it is unlikely that the effect of ethanol-MAK-A on differentiation was mediated by forskolin. Ethanol-MAK-A also induced differentiation in neuroblastoma cells grown in serum-free medium, su~esting that this effect of ethanol-MAK-A was not mediated by serum factors. Another possibility is that ethanol-MAK-A may induce differentiation indirectly by stimulating the release of intracellular factors. The removal of ethanol-MAK-A after the maximum expression of morphological differentiation in neuroblastoma celfs produced different effects, depending upon the presence or the absence of serum. For example, in the presence of serum, the removal of ethanol-MAK-A after 3 days of treatment, caused a rapid degeneration of differentiated neuroblastoma cells within 24 hr. No such degenerative changes were observed if ethanol-MAK-A was not removed during the same period. This suggests that the presence of ethanol-MAK-A in medium containing serum, prevented rapid degeneration of differentiated neuroblastoma cells. In contrast to the observations made in serum, degenerative changes in differentiation neuroblastoma cells was not seen in serumfree medium after the removal of ethanol-MAK-A. On the contrary, when ethanol-MAK-A was removed after 1 or 2 days of treatment, the expression of morphological differentiation in neuroblastoma cells continued to increase in quality and quantity. Even
Ayurvedic agents and dilfferentiation of NB cells
after a week, many differentiated neuroblastoma cells with extensive neurites were observed in this group of cultures. These results suggest that an exposure of neuroblastoma cells to ethanol-MAK-A in serumfree medium for 24 hr or in serum for 3 days, was sufficient to alter the genetic expressions, responsible for maintaining the transformed phenotype of neuroblastoma cells in culture. This alteration then allowed the expression of differentiated phenotype in neuroblastoma cells to be manifested. Previous studies (Prasad, 1975, 1982), have shown that an elevation of the intracellular level of CAMP by prostaglandin E, (PGE,), a stimulator of adenylate cyclase, and by (3-butoxy-4-methoxybenzyl)-2imidazolidinone(R020-1724) an inhibitor of CAMP phosphodiesterase, induced differentiation in neuroblastoma cells similar to that produced by ethanolMAK-A. However, in contrast to MAK-A, these CAMP-stimulating agents are toxic. Further studies are in progress to identify and characterize the differentiating and growth-inhibiting agents that are present in MAK-A. Acknowledgement-This
work
was
suppored
by
the
Lancaster Foundation. REFERENCES
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