To sustain growth and support metabolic requirements, mammals assimilate energy-producing molecules and nutrients from food. These molecules are distributed throughout the body in order to meet the requirements of the internal organs. The various demands of the different organs are to a large extent met by regulatory processes consisting of a complex interaction between hormones, growth factors and cytokines. Normal metabolic activity and partitioning of nutrients between individual organs is affected by a number of events such as stress, a limited supply of nutrients, infection or tumour growth. Since the intestine has the highest metabolic activity of all the internal organs, a tumour will initially compete with the gut for nutrients and energy-providing molecules. The polyamines represent a class of molecules where the demand in the body increases during tumour growth. A tumour can partly obtain the polyamines required to support its growth by up-regulating its own biosynthetic capacity and partly by increasing uptake from the body pool. Rather than limiting the exogenous supply of dietary polyamines we have used another approach to manipulate polyamine pools in mice. When the lectin phytohaemagglutinin is included in the diet, a fully reversible dose-dependent growth of the small intestine occurs leading to an extensive accumulation of polyamines in the intestinal epithelia. This approach of reducing the availability of exogenous polyamines to a growing tumour will be discussed.

Introduction

Our initial observation was that feeding mice on diets containing purified PHA (phytohaemagglutinin), the lectin present in the kidney bean (Phaseolus vulgaris), at an amount of 7 mg of PHA/g, resulted in a prolongation of survival of mice injected intraperitoneally with NHL (non-Hodgkin lymphoma) cells, when compared with mice fed a semi-synthetic control diet based on LA (lactalbumin) as the sole source of protein [1]. Mice fed the LA diet survived for 5–7 days while those fed with PHA had a period of survival of between 15 and 21 days. For the composition of the diets, see [2]. These results will be discussed in the context of the availability of polyamines.

PHA-supplemented diets

Similar to that found in rats [35], when the LA diet was supplemented with PHA, a time-dependent hyperplastic growth of the gastrointestinal tract was also induced in mice [69]. The effect was most marked on the small intestine, where quite dramatic increases in both its wet and dry weight were seen calculated in proportion to total body weight. Owing to the fact that PHA induced a major loss of body lipids, the dry body weight of the mice actually fell below that of control animals pair-fed on the isocaloric LA-based diet [2].

It is evident that, when present in the diet, PHA results in an increased transport of polyamines from the blood into the mucosa of the gut [10,11]. In parallel to the occurrence of hyperplasia, the lectin also appears to stimulate an extensive absorption of nutrients, including amino acids, from the intestinal lumen [12,13]. Interestingly, no marked increase in ornithine decarboxylase activity has been observed in gut tissue in response to the presence of PHA in the diet [10]. It would appear that the increased RNA and protein content of the intestinal mucosa occur simultaneously with the sequestration of polyamines in the tissue [12,13].

PHA stimulates gut hyperplasia

Since it was shown that PHA induces a hyperplastic growth of the gastrointestinal tract within 12 days of feeding the lectin [12,14], it was considered important to study the effects of dietary switch on the resulting hyperplastic response. The proportional dry weight values of the small intestine (percentage dry weight relative to dry body weight) demonstrated that, despite the developing NHL tumour, PHA was still able to induce hyperplasia in all three groups of animals that were fed with the lectin during either part of or for the entire duration of the experiment. Despite the fact that there was a marked degree of gut hyperplasia in mice pre-fed for 3 days before injection of NHL cells and then fed on PHA for a further 8 days (dry organ weight: 363.4±34.6 mg), the weight of the organ attained was 38% lower than that in non-injected PHA-fed mice (580.6±130.3 mg). In comparison, the small intestine in LA-fed mice not injected with tumour cells weighed 334.4±54.2 mg. It therefore appeared that by inducing hyperplasia the lectin prevented the loss in small bowel weight caused by the developing tumour thus more or less maintaining the status quo. In LA-fed tumour-bearing mice, on the other hand, the small intestine weighed 229.0±28.2 mg, which amounted to a loss of 105.4 mg dry weight, being equivalent to a reduction in weight of 31.5%. Taken together, the results suggest that there must exist a major competition between the developing tumour and PHA-induced gut growth for nutrients and growth factors.

Polyamines in NHL tumour cells and gut mucosa

The concentrations of individual polyamines were measured in NHL cells growing as ascites tumours in mice fed on the PHA diet for various time periods. The values were compared with those from mice on the lectin-free diet. The intracellular concentrations of putrescine, spermidine and spermine in NHL cells from mice fed for a total of 11 days on the PHA diet were significantly higher than the values from mice fed the control LA diet. Both these observations and previous results [1517] indicate that the feeding of a PHA diet to mice modulates the intracellular polyamine levels in tumour cells and this seems to be closely associated with growth activity.

After the completion of mitosis, polyamines are redistributed between the two daughter cells. Each then has to synthesize/accumulate sufficient amounts of spermidine and spermine during G1 phase before the cells can enter S-phase [18]. Cells with a short cell cycle duration, such as NHL tumour cells, will have a high demand for polyamines. It is known that, in cancer cells, the intracellular polyamine level is a product of de novo synthesis and a high rate of uptake [18]. The high production of PGE2 (prostaglandin E2) by MCG tumours can be inhibited almost entirely by indomethacin, resulting in a prolonged potential doubling time for tumour growth in vivo. Under these conditions, a decreased cellular content of spermidine was particularly observed [19].

It has been established that the PHA-stimulated hyperplastic growth of the gut in mice [14,15] occurs simultaneously with a sequestration of polyamines in the intestinal mucosa. It is apparent therefore that the two forms of growth activity, i.e. hyperplasia induced by the lectin and the developing tumour, will be in a situation where both will compete for molecules required for growth from a common body pool, this pool including polyamines. This is demonstrated by the fact that the total polyamine content in the tissue of the small intestine in mice injected intraperitoneally with NHL cells and fed a PHA diet for 12 days was approx. 30% lower than that observed in non-injected individuals [15]. The comparative value in LA-fed mice, injected with NHL cells was 13%. It would thus appear that the two forms of growth have roughly the same requirement for polyamines from the available pool.

The results described here indicate a correlation between the great demand exerted by the gut for exogenous polyamines and limited tumour cell growth. It would appear that the build up of polyamines observed in cells from tumours in mice fed for 8 days on the PHA diet is related to cell cycle events, i.e. a large proportion of cells are presumed to be resting in G1 phase. Once the hyperplastic growth of the gut has stabilized at its maximal level, then the demand for extraneous polyamines will be reduced and an increased proportion of tumour cells will be able to obtain the additional polyamines they require from the body pool in order to promote entry into S-phase. It is apparent that there is at least a 3-fold increase in polyamine content in NHL cells before their entry into an active phase of growth. This would in turn suggest that the tumour cells are unable to produce sufficient quantities of polyamines themselves by de novo synthesis through the ornithine decarboxylase system therefore being dependent on access to a supply of exogenous polyamines.

Exogenous polyamines affect tumour growth and the extent of gut hyperplasia

An experiment was performed in order to examine the effect of the addition of exogenous polyamines to the PHA diet on tumour growth, the basic LA diet being virtually polyamine free [20]. Mice were pre-fed on the LA diet for 3 days before subcutaneous injection of NHL tumour cells and then separated into three groups. One group was kept on the same diet, a second group switched to PHA and the third group was switched to a diet of PHA supplemented with putrescine, spermine and spermidine. After a further 11 days, mice were killed and tumours were dissected out. The results shown in Table 1 demonstrate first that a switch to a PHA diet causes a 27% reduction in tumour mass and, secondly, that the addition of polyamines to the PHA diet caused a 44% increase. The addition of exogenous polyamines thus not only reversed the positive effect that the lectin had in reducing tumour mass but also resulted in a tumour which had a 10% higher mass than that which developed in mice fed the lectin-free control LA diet. That PHA induced gut hyperplasia even in the presence of a developing tumour is shown in Table 2, where it can be seen that PHA caused a 32% increase in intestinal mass over a period of 11 days. A further increase of 18% was observed when the PHA diet contained added polyamines. Compared with the mice fed on the LA diet, the polyamine-supplemented PHA diet caused a 55% increase in tissue mass. It is evident from these results that both tumour and hyperplastic growth of the gut were stimulated by the addition of exogenous polyamines to the PHA diet.

Table 1
Changes in NHL tumour mass in mice fed various diets

After 3 days of pre-feeding on the LA diet, three groups of female NMRI mice (five per group) were injected subcutaneously with 2×106 NHL cells and then fed on the diets as indicated. After 8 days, mice were killed and tumours were dissected out, dried and weighed. Results are mean values, and percentage changes relative to LA diet are indicated.

Group… 
Diet LA PHA PHA+polyamines* 
Tumour (mg of wet weight) 373 284 409 
Change (%) – −27 +44 
Group… 
Diet LA PHA PHA+polyamines* 
Tumour (mg of wet weight) 373 284 409 
Change (%) – −27 +44 
*

Diet supplemented with spermidine (0.118 g/kg), spermine (0.027 g/kg) and putrescine (0.026 g/kg).

Table 2
Changes in dry weight of total intestine in mice fed on various diets

After 3 days of pre-feeding on the LA diet, three groups of female NMRI mice (five per group) were injected subcutaneously with 2×106 NHL cells and then fed on the diets as indicated. After 8 days, mice were killed and the total intestine was resected. The tissue was freeze-dried to constant weight. Results are mean values, and percentage changes relative to LA diet are indicated.

Group… 
Diet LA PHA PHA+polyamines* 
Intestine (mg of dry weight) 1583 2083 2456 
Change (%) – +32 +18 
Group… 
Diet LA PHA PHA+polyamines* 
Intestine (mg of dry weight) 1583 2083 2456 
Change (%) – +32 +18 
*

Diet supplemented with spermidine (0.118 g/kg), spermine (0.027 g/kg) and putrescine (0.026 g/kg).

Conclusions

In conclusion, the results presented here add further support to the concept of the importance of the continuous availability of an extraneous source of polyamines to sustain tumour growth. If the supply becomes limited, for example by inducing hyperplasia of the gut, then the rate of tumour growth can be reduced temporarily [21]. The results suggest that by feeding the animal PHA the population of tumour cells becomes at least partially synchronized. Since the S-phase is often a target for anticancer drugs, it is possible that this approach of partial synchronization of the tumour cell population may lead to a higher success rate of chemotherapy.

The observations with respect to alterations in tissue weight and polyamine content promoted by adding a lectin to the diet suggest that inter-organ competition between a tumour and body organs can be used as a tool for the manipulation of tumour growth [21]. In addition to PHA, the supplementation of diets with ML-1 (mistletoe lectin) has also been shown to induce gut hyperplasia [22]. This lectin has proved to be more effective than PHA in reducing tumour growth, since complete ablation of the NHL tumour has been observed in mice [23,24]. It thus appears that, although the two lectins share the same properties, in that when added to the diet they cause hyperplasia of the small intestine, their effects on tumour growth are apparently dissimilar. Taken together, these interesting results may provide novel strategies for the development of new forms of cancer treatment involving the use of plant lectins.

Health Implications of Dietary Amines: A joint COST Action 922 and Biochemical Society Focused Meeting held at Medico-Chirurgical Hall, University of Aberdeen, U.K., 19–21 October 2006. Organized and Edited by H.M. Wallace (Aberdeen, U.K.).

Abbreviations

     
  • LA

    lactalbumin

  •  
  • NHL

    non-Hodgkin lymphoma

  •  
  • PHA

    phytohaemagglutinin

COST 922 is thanked for financial support. Dr Susan Bardocz and Dr Arpad Pusztai, formerly of the Rowett Research Institute, Aberdeen, Scotland, U.K., are acknowledged for initiating this work.

References

References
1
Pryme
I.F.
Bardocz
S.
Pusztai
A.
Cancer Lett.
1994
, vol. 
76
 (pg. 
133
-
137
)
2
Pryme
I.F.
Pusztai
A.
Grant
G.
Bardocz
S.
J. Exp. Ther. Oncol.
1996
, vol. 
1
 (pg. 
273
-
277
)
3
de Oliveira
J.T.A.
Pusztai
A.
Grant
G.
Nutr. Res.
1988
, vol. 
8
 (pg. 
943
-
947
)
4
Bardocz
S.
Brown
D.S.
Grant
G.
Pusztai
A.
Biochim. Biophys. Acta
1990
, vol. 
1034
 (pg. 
46
-
52
)
5
Pusztai
A.
Plant Lectins
1991
Cambridge
Cambridge University Press
6
Pryme
I.F.
Pusztai
A.
Bardocz
S.
Int. J. Oncol.
1994
, vol. 
5
 (pg. 
1105
-
1107
)
7
Bardocz
S.
Grant
G.
Duguid
T.J.
Brown
D.S.
Pusztai
A.
Pryme
I.F.
Int. J. Oncol.
1994
, vol. 
5
 (pg. 
1369
-
1374
)
8
Pryme
I.F.
Pusztai
A.
Bardocz
S.
Ewen
S.W.B.
Histol. Histopathol.
1998
, vol. 
13
 (pg. 
575
-
583
)
9
Pryme
I.F.
Pusztai
A.
Bardocz
S.
Ewen
S.W.B.
Johnson
I.T.
Fenwick
G.R.
Dietary Anticarcinogens and Antimutagens: Chemical and Biological Aspects
2000
Gateshead
Athenaeum Press
(pg. 
350
-
354
)
10
Bardocz
S.
Grant
G.
Brown
D.S.
Ewen
S.W.B.
Nevison
I.
Pusztai
A.
Digestion
1990
, vol. 
46
 
Suppl.
(pg. 
360
-
366
)
11
Pusztai
A.
Grant
G.
Williams
L.M.
Brown
D.S.
Ewen
S.W.B.
Bardocz
S.
Med. Sci. Res.
1989
, vol. 
17
 (pg. 
215
-
217
)
12
Bardocz
S.
Grant
G.
Duguid
T.J.
Brown
D.S.
Sakhri
M.
Pusztai
A.
Pryme
I.F.
Mayer
D.
Way
K.
Med. Sci. Res.
1994
, vol. 
22
 (pg. 
101
-
103
)
13
Bardocz
S.
Grant
G.
Duguid
T.J.
Brown
D.S.
Pusztai
A.
Pryme
I.F.
Int. J. Oncol.
1994
, vol. 
5
 (pg. 
1369
-
1374
)
14
Pryme
I.F.
Grant
G.
Pusztai
A.
Bardocz
S.
Bardocz
S.
White
A.
Polyamines in Health and Nutrition
1999
Norwell
Kluwer Academic Publishers
(pg. 
283
-
291
)
15
Pryme
I.F.
Bardocz
S.
Grant
G.
Duiguid
T.J.
Brown
D.S.
Pusztai
A.
Cancer Lett.
1995
, vol. 
93
 (pg. 
233
-
237
)
16
Bardocz
S.
Grant
G.
Duguid
T.J.
Brown
D.S.
Pusztai
A.
Pryme
I.F.
Cancer Lett.
1997
, vol. 
121
 (pg. 
25
-
29
)
17
Bardocz
S.
Grant
G.
Ewen
S.W.B.
Pryme
I.F.
Pusztai
A.
Bardocz
S.
Pfüller
U.
Pusztai
A.
Effects of Antinutrients on the Nutritional Value of Legume Diets (COST 98), volume 5
1998
Luxembourg
European Commission Publications
(pg. 
208
-
214
)
18
Seiler
N.
Delgros
J.G.
Moulinoux
J.P.
Int. J. Biochem. Cell Biol.
1996
, vol. 
28
 (pg. 
843
-
861
)
19
Lonnroth
C.
Svaninger
G.
Gelin
J.
Cahlin
C.
Iressjo
B.M.
Cvetvkovska
E.
Edström
S.
Andersson
M.
Svanberg
E.
Lundholm
K.
Int. J. Oncol.
1995
, vol. 
7
 (pg. 
1405
-
1413
)
20
Pryme
I.F.
Pusztai
A.
Ewen
S.W.B.
Bardocz
S.
Morgan
D.M.L.
White
A.
Sanchez-Jimenez
F.
Bardocz
S.
Biogenically Active Amines in Food (COST 917), volume 4
2000
Luxembourg
European Commission Publications
(pg. 
167
-
172
)
21
Pryme
I.F.
Bardocz
S.
Eur. J. Gastroenterol. Hepatol.
2001
, vol. 
13
 (pg. 
1
-
6
)
22
Pryme
I.F.
Bardocz
S.
Pusztai
A.
Ewen
S.W.B.
Histol. Histopathol.
2002
, vol. 
17
 (pg. 
261
-
271
)
23
Pryme
I.F.
Bardocz
S.
Pusztai
A.
Ewen
S.W.B.
Pfüller
U.
Cancer Detect. Prev.
2004
, vol. 
28
 (pg. 
52
-
56
)
24
Pryme
I.F.
Bardocz
S.
Pusztai
A.
Ewen
S.W.B.
Histol. Histopathol.
2006
, vol. 
21
 (pg. 
285
-
299
)