Uriel Bachrach

Polyamines and cancer: Minireview article

Summary.The naturally occurring polyamines, spermine, spermidine and the diamine putrescine are widespread in nature. They have been implicated in growth and differentiation processes. Polyamines accumulate in cancerous tissues and their concentration is elevated in body fluids of cancer patients. Assays of urinary and blood polyamines have been used to detect cancer and to determine the success of therapy. Drugs which inhibit the synthesis of polyamines can prevent cancer and may also be used for therapeutic purposes. Ornithine decarboxylase, which catalyzes the rate limiting step in polyamine synthesis, can serve as a marker of proliferation. Recently, a new in vitro chemosensitivity test, based on the disappearance of ornithine decarboxylase in drug-treated cancer cells has been developed. The increasing interest in polyamines and their physiological functions may lead to a more extensive application of these compounds or their derivatives in cancer diagnosis and treatment.

Ornithine decarboxylase activity in rapidly proliferating plant cells

Polyamines are widely distributed in nature, but their precise role in cellular processes is not fully understood. Polyamines are associated with cell proliferation, tissue regeneration and malignancy [l-3]. Most of the information on the biosynthetic pathways of putrescine, spermlne and spermidine, their regulation and possible sites of action has been obtained from studies with micro-organisms or mammalian cells. In mammalian cells, putrescine is synthesized from Lornithine by Lornithine decarboxylase (ODC EC 4.11.17) [2]. In plants, however, it is commonly accepted that putrescine is formed from Larginine by Larginine decarboxylase (ADC EC 4.11.19), via the intermediate agmatine. The activity of ODC in plants is usually found to be much lower than that of ADC [4-71, and ODC has therefore been claimed to be of little significance in the formation of putrescine in plants. We felt that in plant cells such an essential biosynthetic pathway of putrescine should not differ from that in mammalian cells and therefore decided to study ODC activity in plant systems. We chose to search for ODC activity in two plant systems of rapidly proliferating cells the XD cell line of tobacco in suspension culture and tomato ovaries before and after pollination. After subculture of XD cells there is usually a lag period of a few days, after which the culture enters an exponential phase, lasting for several generations [8]. The culture then enters a stationary phase. In tomato ovaries before pollination there is some cell division; after pollination there is very intense cell division, which lasts for

The specific anti-cancer activity of green tea (-)-epigallocatechin-3-gallate (EGCG).

Summary. The effect of the green tea polyphenol-(−)epigallocatechin-3-gallate (EGCG) was tested in cultures of normal and transformed NIH-pATMras fibroblasts. In this system transformation can be induced at will by the addition of dexamethasone, which induces the expression of H-ras by activating the mammary tumor virus long terminal repeat (MMTV-LTR) promoter. This facilitates a reliable comparison of the susceptibility of normal and transformed cells to EGCG. It has been shown that EGCG inhibited the growth of transformed but not of the normal fibroblasts. In an attempt to elucidate the mode of the preferential inhibitory activity of EGCG, its effect on growth promoting factors has been examined. The level of ornithine decarboxylase (ODC, EC 4.1.1.17), which is a signal for cellular proliferation, was reduced by EGCG in the transformed but not in the normal cells. EGCG also showed strong inhibition of tyrosine kinase and mitogen-activated protein kinase (MAPK) activities, without affecting the kinases in the normal cells. Similarly, EGCG also preferentially decreased the levels of the oncogenes Ras and Jun in transformed cell. EGCG preferentially induced apoptosis in the transformed fibroblasts. In vitro chemosensitivity tests demonstrated that EGCG inhibited the proliferation of leukemic cells. These findings suggest that EGCG has a therapeutic potential in the combat against cancer.

Cytotoxic effect of oxidized spermine on Ehrlich ascites cells

Abstract Spermine, after oxidation by highly purified serum amine oxidase (amine: O2 oxidoreductase (deaminating)), inhibited the multiplication of Ehrlich ascites cells. The oxidation product, oxidized spermine, was separated from the amine oxidase by gel filtration and its toxicity verified. The related triamines, spermidine and diaminodipropylamine, were also oxidized by purified serum amine oxidase, giving rise to cytotoxic products. The polyamines in the absence of amine oxidase did not affect the multiplication of the Ehrlich ascites cells, whereas purified amine oxidase alone exhibited a slight cytotoxic effect. Oxidized [14C]spermine was rapidly taken up by Ehrlich ascites cells on incubation at 37°. The incorporation of radioactive valine, thymidine or uracil, into the corresponding cellular macromolecules, was markedly reduced after treating Ehrlich ascites cells with oxidized spermine for 30 min at 37°.

Naturally occurring polyamines: interaction with macromolecules.

The naturally occurring polyamines, spermine [NH2(CH2)3NH(CH2)4NH(CH2)3NH2] and spermidine [NH2(CH2)3NH(CH2)4NH2], as well as the diamine putrescine [NH2(CH2)4NH2], are widely spread in nature. They occur in plants, micro-organisms and animal tissues and fulfil many important physiological functions. Due to their cationic nature they interact with negatively charged macromolecules such nucleic acids, phospholipids and proteins. This ionic interaction, which is reversible, leads to the stabilization of DNA, tRNA, membranes and some proteins. Early studies demonstrated that polyamines stimulate the growth of pro- and eukaryotic cells and that they play an important role in carcinogenesis and in malignant transformation processes. As a result of these studies various inhibitors of polyamine biosynthesis have been synthesized and are used to combat cancer and parasitic diseases (e.g., African sleeping sickness).

Cancer therapy and prevention by green tea: role of ornithine decarboxylase.

Summary. Green tea which is widely consumed in China, Japan and India, contains polyphenolic compounds, which account for 30% of the dry weight of the leaves. Most of the polyphenols are flavanols, of which (−)-epigallocatechin-3-gallate (EGCG) is most abundant. Epidemiological studies revealed that the incidences of stomach and prostate cancers are the lowest in the world among a population that consumes green tea on a regular basis. It has also been reported that the quantity of green tea consumed, plays an important role in reducing cancer risk and in delaying cancer outbreak and recurrence. Various systems were used to confirm anti-cancer activities of green tea and/or EGCG. These included experimental animals in which cancer was induced chemically. Cultured cells transformed chemically or by oncogenes were also used. These studies clearly demonstrated that green tea or EGCG have anticancer and cancer preventive properties. The mechanisms of these activities have also been studied in details. It has been shown that green tea and its active components interfere with signal transduction pathways. Thus the activities of various protein kinases are inhibited, the expression of nuclear proto-oncogenes declines and the activity of ornithine decarboxylase (ODC) is reduced. ODC, which catalyzes the rate-limiting step in the biosynthesis of polyamines is closely linked with cellular proliferation and carcinogenesis. Inhibitors of ODC, like α-difluoromethylornithine (DFMO) have long been used for cancer prevention and therapy. It has been suggested that polyamine depletion by green tea could offer one explanation for its anti-cancer activities.

Role of polyamines in mediating malignant transformation and oncogene expression

Previous studies have demonstrated that polyamines accumulate in cancer cells and that overproduction of ornithine decarboxylase (ODC), which catalyzes polyamine synthesis, elicits the acquisition of the transformed phenotype. However, it was not clear whether the expression of ODC and the accumulation of polyamines are only innocent by-products of the transformation process. In this study we confirm previous findings what polyamines can trigger the transformation of immortalized cultured cells. In addition to NIH 3T3 fibroblasts, studied previously, rat kidney epithelial cells or fibroblasts also grew in soft agar in the presence of polyamines. It has also been demonstrated that spermidine, preferentially stimulated the transcription and the expression of c-myc while those of c-fos were preferentially stimulated by putrescine. These findings suggest that the effect of polyamines on cellular transformation, could be explained, at least partially, by stimulation of proto-oncogene expression.

Enzymic assay for spermine and spermidine

Abstract This paper describes a sensitive enzymic micromethod for the quantitative assay of spermine and spermidine. The method is based on the oxidation of the polyamines by serum amine oxidase. The aminoaldehydes produced during the oxidation are assayed by N -methyl-2-benzothiazolone hydrazone hydrochloride, at 660 mμ. The molar absorbancy index for spermine is 12.5 × 10 3 liter mole −1 cm −1 and for spermidine 6.25 × 10 3 liter mole −1 cm −1 . When both spermine and spermidine are present in the biological material the value of the “combined polyamines” is obtained by the enzymic method. Spermidine is assayed by another method that uses dried Serratia marcescens cells. This permits the calculation of spermine by subtracting the amount of spermidine from that of the “combined polyamines.” The enzymic method has been applied to extracts from yeast, bacteria, and mouse liver.

Induction of ornithine decarboxylase in glioma and neuroblastoma cells.

Ornithine decarboxylase (ODC, Lornithine carboxylyase; EC 4.1.17), catalyzes the conversion of ornithine to the diamine, putrescine. This reaction is the ratelimiting step in the synthesis of putrescine and the naturally occurring polyamines, spermidine and spermine which have been linked to many processes crucial for cell growth, division and differentiation [ 1,2] . Recent studies indicated the presence of polyamines in neural tissues [3,4] and in cultures of mouse neuroblastoma cells [5,6]. This paper deals with the induction of ODC in C6-BU-1 giioma and N115 neuroblastoma cells by fresh medium added to confluent cultures. A similar induction of ODC by fresh medium has been reported for other cell lines [ 7121.

Polyamines induce malignant transformation in cultured NIH 3T3 fibroblasts

Previous studies have demonstrated that polyamines accumulate in cancer cells and that overproduction of ornithine decarboxylase (ODC), which catalyzes polyamine synthesis, elicits the acquisition of the transformed phenotype. However, it was not clear whether the overexpression of ODC and the accumulation of polyamines are only innocent by-products of the transformation process. In this study we demonstrate that polyamines as such, may play a crucial role in malignant transformation. The system used consisted of NIH 3T3 fibroblasts transfected with a construct (pATMras) in which Ha-ras was under the transcriptional control of the mouse mammary tumor virus long terminal repeat (MMTV-LTR) promoter (MMTVras cells). Dexamethasone, which activates the promoter, triggered phenotypic transformation. This was accompanied by an increase in ODC activity and polyamine accumulation. Cells, thus transformed, grew in soft agar and formed typical foci. alpha-Difluoromethylornithine (DFMO), which blocks polyamine synthesis, inhibited the dexamethasone-enhanced transformation. This inhibition was reversed by polyamines. Polyamines caused transformation of MMTVras cells in the absence of dexamethasone. Under these conditions, cells became anchorage independent. This phenomenon is not explained by the leakiness of ras, since normal, immortalized NIH 3T3 fibroblasts, also grew in soft agar in the presence of polyamines. Taken together, these observations suggest that polyamines may stimulate malignant transformation of immortalized cells, in cooperation with other factors, such as oncogenes or genetic defects.