Promotion Stage Of Carcinogenesis Research Articles

Stimulation of DNA synthesis by tumor promoters in primary rat hepatocytes is not mediated by arachidonic acid metabolites

Studies in vivo using inhibitors of eicosanoid synthesis suggested that prostaglandins may play a role in mediating tumor promotion in liver by agents such as phenobarbital (PB). However, it is not clear whether any stimulation of arachidonic acid metabolism/prostaglandin formation results directly from the action of tumor promoters on hepatocytes or indirectly from effects of promoters on Kupffer cells or other non-hepatocytes. Our laboratory has been utilizing relatively pure populations of rat hepatocytes under the defined conditions of primary cultures, to investigate growth-stimulatory actions of tumor promoters, an important element in the promotion stage of carcinogenesis. It has been shown that most if not all liver tumor promoters tested stimulate hepatocyte DNA synthesis when added in combination with factors such as EGF, insulin, and glucocorticoid. In the present study, we sought evidence for a role of prostaglandins (PGs) in the direct growth-stimulatory actions of tumor promoters on hepatocytes. PGE(2), PGF(2 alpha), and PGD(2) cause concentration-dependent stimulation of hepatocyte DNA synthesis, while arachidonic acid was without any effect. PGE(2) and PGF(2 alpha) required the presence of dexamethasone to exert significant effects. These PGs did not further augment the stimulatory effect of EGF. In contrast, PGD(2) stimulated DNA synthesis in the presence or absence of insulin, dexamethasone, or EGF. The effect of tumor promoters on arachidonic acid metabolism, as measured by [(3)H]arachidonic acid release and PGE(2) production, was determined. The phorbol ester TPA significantly increased [(3)H]arachidonic acid release as well as PGE(2) formation in hepatocytes in line with known effects in other cell types. However, liver tumor promoters phenobarbital (PB), alpha-hexachlorocycohexane (HCH), 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), and pregnenolone-16 alpha-carbonitrile (PCN) were without effects. Finally, inhibitors of arachidonic acid metabolism were tested for effects on the ability of TPA or liver tumor promoters to stimulate DNA synthesis by direct action on cultured hepatocytes. In all cases, lack of selective inhibition was observed. Taken together, the results show that while prostaglandins may directly stimulate DNA synthesis in hepatocytes, they are unlikely to mediate the direct growth-stimulatory actions of liver tumor promoters.

Effect of wheat bran fiber on the development of mammary tumors in female intact and ovariectomized rats treated with 7,12-dimethylbenz(a)anthracene and in mice with spontaneously developing mammary tumors.

We examined the effect of consumption of graded increases of dietary fiber (soft white wheat bran) on the development of mammary gland carcinomas in intact female Sprague-Dawley rats during the promotion stage of carcinogenesis, induced with 7,12-dimethylbenz(a)anthracene (DMBA). The percent of rats with mammary carcinomas, the total number of mammary carcinomas and the mean number of mammary carcinomas per rat were reduced significantly at all fiber levels examined compared to rats fed a control diet. Inclusion of 9.6% fiber in the diets of ovariectomized rats that had been treated with a single i.v. dose of 2.5 mg DMBA/100 g body weight 2 weeks prior to removal of the ovaries resulted in a significant decrease of carcinomatous and benign mammary tumors compared to ovariectomized rats fed a control diet. Development of spontaneous mammary carcinomas in virgin C3H/HeOuJ female mice and growth of a transplantable mammary gland tumor in such mice were reduced by inclusion of 9.6% fiber in the diet, a reduction that was significant or just barely missed significance, depending on the source of the fiber. Our observations provide evidence that inclusion of soft white wheat bran in the diet is effective in the suppression of mammary gland tumorigenesis in an array of experimental animal models.

Hormone Dependence of Mammary Tumors Induced in Rats by Intraperitoneal NMU Injection

The purpose of this work was to determine the hormone dependence of mammary tumors induced in Sprague-Dawley rats by three intraperitoneal injections of N-nitroso-N-methylurea at 50, 80, and 110 days of age. Two experimental designs were carried out: (a) Ten days before the first NMU injection, 130 rats were divided into 13 batches and randomly assigned to the following treatments: control, ovariectomy (OVX), tamoxifen (TAM), bromocriptine (BROM), haloperidol (HAL), estradiol (E2), progesterone (Pg), OVX % BROM, TAM % BROM, OVX % HAL, TAM % HAL, OVX % TAM, andc E2% BROM. After 150 days of treatment the following growth parameters were determined: latency period (LP), mean tumor number per rat (n/t), and tumor incidence (TI). LP was significantly increased (p < 0.05) only by Pg and TAM % BROM. The n/t was significantly decreased (p < 0.05) by all treatments except HAL. TI was significantly reduced by OVX, TAM, BROM, and their combinations, (b) Rats bearing ip-NMU-induced mammary tumors were divided into 7 batches and assigned to the following treatments: control, OVX, TAM, BROM, HAL, OVX % BROM, and TAM % BROM. Tumor growth was assessed up to 60 days of treatment; only OVX, TAM and their combination with BROM were able to produce tumor regression. These results support the essential role of E2and prolactin in the promotion stage of carcinogenesis. However, for established tumors, growth becomes more independent from hormone influence, in particular from prolactin deprivation. We conclude that this model seems suitable for studying the mechanisms underlying the evasion of hormonal control of tumor growth.

Quantitative analysis of changes in cell proliferation and apoptosis during preneoplastic and neoplastic stages of hepatocarcinogenesis in rat.

In situ markers for quantitative analysis of cell proliferation and apoptotic cell death have been used to evaluate both the proliferation level and net growth potential of preneoplastic nodules and malignant tumor tissues from rats experimentally induced for hepatocarcinogenesis by the dietary administration of 2-acetylaminofluorene. The findings show that although tumors have a much higher level of cell proliferation than preneoplastic liver nodules, the nodules have a higher potential for net growth when apoptosis is taken into account. These results support a role for a decrease in apoptosis during the promotion stage of carcinogenesis.

Effect of diverse tumor promoters on the expression of gap-junctional proteins connexin (Cx) 26, Cx31.1, and Cx43 in SENCAR mouse epidermis

The inhibition of gap-junctional intercellular communication (GJIC) between initiated and surrounding normal cells by tumor promoters is believed to be important in the promotion stage of carcinogenesis. Therefore, we examined the effect of skin-tumor promoters on the expression of the gap-junctional proteins connexin (Cx) 26, Cx43, and Cx31.1 in SENCAR mouse skin. Animals were treated with 12-0-tetradecanoylphorbol-13-acetate (TPA) (8.3 nmol), okadaic acid (OA) (2.5 nmol), chrysarobin (220 nmol), or benzoyl peroxide (BzPo) (83 micromol). Northern blot and immunofluorescence analyses revealed that keratinocytes in adult mouse skin expressed Cx31.1 and Cx43 but not Cx26. All four of the skin-tumor promoters switched on the Cx26 gene, transiently increased expression of Cx43, and significantly inhibited the expression of Cx31.1. The time courses for changes in Cx26, Cx3l. 1, and Cx43 mRNA levels coincided in most cases and in general corresponded well to the time-response curves for hyperplastic changes in mouse skin. The peaks of Cx26 and Cx43 expression and Cx31.1 inhibition appeared 12 h after TPA application and 24 h after OA and chrysarobin application. BzPo elevated the levels of Cx26 and Cx43 transcripts later (peak at 2-4 d). In tumor promoter-treated skin, Cx26 and Cx43 plaques were on the plasma membrane of most keratinocytes. Cx31.1 staining was much weaker than in untreated epidermis. Thus, tumor promoters induce a large change in the expression of several Cxs, which in turn may affect both the level of GJIC and the sensitivity of GJlC to regulatory factors.

Effects of protease inhibitors on levels of proteolytic activity in normal and premalignant cells and tissues.

Our studies utilizing different types of protease inhibitors as anticarcinogenic agents in in vivo and in vitro systems have recently been reviewed. These studies suggest that the protease inhibitors which prevent carcinogenesis affect processes in the early stages of carcinogenesis, although they can be effective at long time periods after carcinogen exposure in both in vitro and in vivo systems. While there is strong evidence that these protease inhibitors can affect both the initiation and promotion stages of carcinogenesis, they have no effect on already transformed cells. Our results have suggested that the first event in carcinogenesis is a high frequency epigenetic event and that a later event, presumably genetic, leads to the malignant state. Protease inhibitors appear capable of reversing the initiating event, presumably by stopping an ongoing cellular process begun by carcinogen exposure. The major lines of investigation on the mechanism of the protease inhibitor suppression of carcinogenesis relate to the ability of anticarcinogenic protease inhibitors to affect the expression of certain oncogenes, and the levels of certain types of proteolytic activities. The anticarcinogenic protease inhibitors have no observable effects on normal cells, but can reverse carcinogen-induced cellular changes for several different end-points studied. The most direct method of determining the mechanism of action of the anticarcinogenic protease inhibitors is to identify and characterize the proteases with which they interact. In the cells of the in vivo and in vitro systems in which protease inhibitors can prevent carcinogenesis, only a few proteases have been observed to interact with the anticarcinogenic protease inhibitors. Proteases have been identified by both substrate hydrolysis and affinity chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of chemopreventive effect of dietary selenium-rich egg on mouse skin tumor induced by 2'-(4-nitrophenoxy)oxirane and 12-O-tetradecanoylphorbol-13-acetate.

Chemoprotective effect of dietary selenium (as sodium selenite or as Se-rich egg) on mouse skin tumor induced by topical application of 2'-(4-nitrophenoxy)oxirane (NPO) as tumor initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as tumor promoter was evaluated in relation to the dietary source and levels of selenium. Selenium supplementation (0.3 p.p.m.) to the basal diet (0.07 p.p.m. Se) as sodium selenite or as Se-rich egg brought about a 40 or 37% reduction respectively, in the incidence of papilloma formation at 12 weeks after NPO treatment. Tumor yield (number of papillomas per mouse) at 14 weeks after NPO treatment in the basal diet group, basal diet supplemented with 0.3 p.p.m. Se as sodium selenite group and basal diet supplemented with 0.3 p.p.m. Se as Se-rich egg group were 7.5 +/- 2.1, 2.7 +/- 2.3 and 4.1 +/- 3.5 respectively. Dietary supplementation of 1.0 p.p.m. See as Se-rich egg to the basal diet reduced the incidence and the multiplicity of papillomas during the early phase of promotion (11 weeks) but its antitumor activity decreased thereafter, indicating that the accumulation of tissue selenium above the saturated level may not be beneficial. Selenium concentrations in blood, liver and skin tissue of mice in basal diet group (0.33 +/- 0.02, 0.54 +/- 0.10 and 0.21 +/- 0.03 p.p.m. respectively) increased significantly ( P < 0.05) by the supplementation of 0.3 p.p.m. Se as selenite (0.58 +/- 0.02, 1.17 +/- 0.10 and 0.31 +/- 0.05 p.p.m. respectively), 0.3 p.p.m. Se as Se-rich egg (0.59 +/- 0.02, 1.25 +/- 0.11 and 0.33 +/- 0.06 p.p.m. respectively) and 1.0 p.p.m. Se as Se-rich egg (1.20 +/- 0.05, 2.32 +/- 0.28 and 0.51 +/- 0.01 p.p.m. respectively). Glutathione peroxidase activity in blood of mice of the basal diet group (2.4 +/- 0.4 EU/mg) increased significantly (P < 0.05) by dietary selenium supplementation (3.8 +/- 0.6 EU/mg in 0.3 p.p.m. Selenite; 3.7 +/- 0.8 EU/mg in 0.3 p.p.m. Se as Se-rich egg; 4.9 +/- 0.9 EU/mg in 1/0 p.p.m. Se as Se-rich egg) and the enzyme activities in liver and skin tissue also increased by 0.3 p.p.m. Se (as selenite or Se-rich egg) supplementation, but no further increases in their activities were obtained by 1.0 p.p.m. Se (as Se-rich egg). It is, therefore, concluded that a moderate level of dietary selenium (0.3 p.p.m.) has an efficient chemopreventive activity at the promotional stage of carcinogenesis and that dietary selenium rich egg as well as dietary selenite exerted antitumor activity.

Dietary supplementation with pectin and guar gum on 1,2-dimethylhydrazine-induced colon carcinogenesis in rats

The effect of dietary supplementation with pectin and/or guar gum on 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis was studied using 120 male Sprague-Dawley rats. The rats were given a weekly injection of DMH for 8 weeks and were maintained on a basal fiber-free diet supplemented with 5% cellulose. The rats were then subdivided into four groups and kept on the basal fiber-free diet supplemented with either no fiber, 10% pectin, 10% guar gum or a combination of 5% pectin/5% guar gum for a period of 24 weeks. The 8 weeks of DMH administration were defined as the initiation stage of carcinogenesis and the next 24 weeks were defined as the promotional stage of carcinogenesis. Food and water were available ad libitum. The rats were killed 32 weeks after the start of the experiment and tumor incidence, location and frequency in the colon were determined. Other parameters measured were body weight and caloric intake. Dietary fiber supplementation with 10% pectin or with 10% guar gum but not with the combination of 5% pectin/5% guar gum (fed during the promotional stage of carcinogenesis), was found to suppress colon cancer incidence to a significant extent.

Hypomethylation of ras oncogenes in chemically induced and spontaneous B6C3F1 mouse liver tumors.

The male hybrid B6C3F1 mouse exhibits a 30% spontaneous hepatoma incidence, and both males and females of this strain are sensitive to chemical induction of liver tumors. The Ha-ras, Ki-ras, and myc oncogenes have been implicated in a variety of solid tumors. Specifically, Ha- and, less frequently Ki-ras have been reported to be activated in B6C3F1 mouse liver tumors, and such activated oncogenes frequently contain a particular point mutation. In light of indications that the transforming capacity of some oncogenes is directly related to the level of the gene product, we hypothesized that transcriptional control of Ha-ras, Ki-ras, and myc is compromised in B6C3F1 mouse liver tumors. A positive correlation has been established between gene expression and hypomethylation. Therefore, the methylation states of these genes were examined in spontaneous liver tumors and in tumors induced by two diverse hepatocarcinogens: phenobarbital and chloroform. Ha-ras was found to be hypomethylated in all tumors examined, whereas Ki-ras was sometimes hypomethylated; such hypomethylation might play a role in the promotion stage of carcinogenesis. The methylation state of myc was unaltered, although this gene appeared to be amplified in tumors. These results suggest that a component of the mechanism by which these oncogenes are activated in B6C3F1 mouse liver tumors involves loss of stringent control of expression, via hypomethylation of the ras oncogenes and, possibly, amplification of myc. These results support the assertion that tumors induced by different classes of carcinogens or arising spontaneously share common biochemical pathways of oncogene activation during tumorigenesis.

Phenotype of preneoplastic and neoplastic liver lesions during spontaneous liver carcinogenesis of LEC rats

The incidence and phenotype of preneoplastic and neoplastic liver lesions appearing in LEC rats after recovery from severe hereditary hepatitis were studied in comparison with the liver lesions appearing in chemical liver carcinogenesis. The livers of 168 rats (90 male, 78 female) were stained for seven histochemical markers at different time periods from the 20th week to the 122nd week of life. Glucose-6-phosphatase (G6Pase), adenosine triphosphatase (ATPase) and non-specific esterase (ES) were used as negative markers. Gamma-glutamyltransferase (GGT), glutathione S-transferase placental form (GSTP), esterase isozyme L-1 (L1) and alpha-fetoprotein (AFP) were used as positive markers. The study on the incidence of liver lesions in the LEC rats revealed sequential development of liver foci, nodules and hepatocellular carcinomas (HCCs) similar to those seen in chemically induced liver carcinogenesis. These lesions appeared earlier and more frequently in male LEC rats than in female ones, suggesting the importance of hormonal environment in spontaneous HCC development. The histochemical analysis of spontaneous liver lesions in LEC rats showed that GSTP was the most reliable positive marker as previously reported in chemical liver carcinogenesis. There was no essential difference in the expression of the markers in spontaneous and chemically induced liver lesions except for L1, which is considered to be related to xenobiotic metabolism. The results of this study suggest that both spontaneous and chemically induced liver cancer may develop by passing through phenotypically similar preneoplastic processes. In addition, the LEC rat uniquely showed chronic liver damage (hepatocyte death and regeneration) at the promotion stage of carcinogenesis. Such a natural history of HCC development in LEC rats is similar to that of human HCC which is frequently associated with chronic liver damage. Thus, the LEC rat provides a useful model for studying the process and underlying mechanisms of human liver cancer development.

Diet and carcinogenesis: historical perspectives.

Some 40-50 years ago, experiments with animals began to provide conclusive evidence that diet can significantly influence carcinogenesis. Underfeeding or severe restriction of caloric intake to between one-half and two-thirds of normal had a general inhibitory effect on development of tumors of various types. On the other hand, high fat diets were found to enhance tumor yields relative to low fat diets. The effect of dietary fat was shown to be independent of caloric intake and was more selective for certain types of tumors. Both dietary fat and caloric restriction appear to exert their effects mainly at the promotional stage of carcinogenesis, although the exact mechanisms are not known. Interest in the role of diet in carcinogenesis has been revived in recent years by epidemiologic data that show correlations between certain dietary constituents, such as dietary fat, and cancer incidence at particular sites in the body and mortality. These epidemiologic observations have, in turn, led to an increase in research on effects of diet on carcinogenesis in animal models.

β-Carotene reduces sister chromatid exchanges induced by chemical carcinogens in mouse mammary cells in organ culture

Present studies in the mammary epithelial cell transformation model in organ culture showed that presence of β-carotene during the 24 hr treatment (initiation stage) of the glands with the carcinogens, 7,12-dimethylbenz[a]anthracene (DMBA), N-nitrosodiethylamine (DENA) and N-methylnitrosourea (MNU), caused a highly significant (P < 0.001-0.01) reduction of SCE induced by the same carcinogens. In contrast, 4-hydroxyphenyl retinamide (4-HPR) which is known to act at the promotional stage of carcinogenesis did not show any significant reduction of SCE. Thus findings suggest that β-carotene can modify the DNA damaging effect of the carcinogens and thereby may also prevent the initiation of the carcinogenic process.

Nutrition and Cancer

As a biomedical area, the association between diet and cancer is at about the same stage of development as was diet and atherosclerotic vascular disease 20 years ago. We have tantalizing epidemiologic observations, which, along with dietary studies utilizing experimental animals, suggest that a variety of dietary components may modulate the transformation and promotion stages of carcinogenesis. Among these are the energy nutrients, several vitamins and minerals, and nonnutrient substances, which are both natural and inadvertent constituents of the food supply. This diversity of agents, as well as the observations that many individual factors are site-specific in their effect on target organs, makes this a complex field indeed. The first chapter of this book presents an overview of the entire field and is a good summary of the scope of the problem. The subsequent sections are based on the several categories of individual nutrients. Each furnishes an extensive and technical,

Neoplastic growth of carcinogen-treated mammary transplants as influenced by fat intake of donor and host

Female Wistar-Furth rats were fed either a high-fat (HF) or a low fat (LF) diet from weaning. The HF and LF diets contained 20% and 0.5% corn oil, respectively. Mammary gland explants from 50-day-old rats of both dietary groups were exposed to dimethylbenz[ a]anthracene in organ culture before grafting to HF- or LF-hosts. A total of 4 groups were involved: LF → LF, HF → LF, LF → HF, and HF → HF, where the designations before and after the arrow describe the dietary treatment of the donor and host, respectively. Final tumor incidences were as follows: 28%, 20%, 72% and 76%, presented in the order of the above 4 groups. Tumors also appeared earlier in the HF-hosts. Results of this experiment indicate that regardless of the nutritional status of the donor, it was the fat intake of the host that governed the subsequent neoplastic growth of transformed cells, suggesting that the action of fat is primarily exerted at the promotional stage of carcinogenesis.

Carcinogenesis Induced by 7,12-Dimethylbenz[a]anthracene in C3H-A vyfB Mice: Influence of Different Dietary Fats

The effect of a diet containing either sunflower-seed oil (polyunsaturated fat diet) or tallow (saturated fat diet) on 7,12-dimethylbenz[a]anthracene (DMBA)-induced carcinogenesis in C3H-A vyfB mice was examined. After receiving either diet for 28 days, some of the mice were given an intragastric dose of 5 mg DMBA. To identify the stage of carcinogenesis that might be influenced by dietary fat, the diets of half of the mice were then interchanged so that those previously fed the saturated fat diet were fed the polyunsaturated fat diet and vice versa. The cumulative incidence of tumor-bearing mice was significantly greater among the females fed the polyunsaturated fat diet compared to those fed the saturated fat diet. This enhancement of carcinogenesis was observed only when the mice were fed the polyunsaturated fat diet after DMBA administration. Similar trends were observed in the male mice, but these mice developed fewer tumors and none of the differences between the tumor incidences were statistically significant. The most common sites for tumors in the male mice were the liver, lungs, and skin, whereas those for tumors in the females were the mammary glands and ovaries. The differences in tumor incidence suggest that carcinogenesis was enhanced by the polyunsaturated fat diet during the promotion stage of carcinogenesis.

Effect of dietary polyunsaturated fat on the growth of a transplantable adenocarcinoma in C3HAvyfB mice.

Weaning male and female C3HAvyfB mice were fed a low-fat (4.5%) diet until they were 60-70 days of age when they were fed high-fat (18.6%) diets containing either sunflower-seed oil (polyunsaturated fat diet) or tallow (saturated fat diet). After receiving either of the high-fat diets for 4 weeks, each mouse received an inoculum of approximately 1,700 single cells from a transplantable mammary adenocarcinoma. The cumulative incidence of tumor-bearing mice was significantly greater among both males and females fed the polyunsaturated fat diet than among males and females fed the saturated fat diet. The mean times elapsed before palpable tumors developed were less when mice were fed the polyunsaturated fat diet than when mice were fed the saturated fat diet, but these differences were not statistically significant. The cumulative incidence of tumor-bearing mice was also significantly greater among females than males. The results supported the suggestion from previous work in this laboratory that the polyunsaturated fat diet exerts its effect on the promotional stage of carcinogenesis rather than on the initial event of neoplastic transformation.

Minireview possible roles of dietary fats in carcinogenesis

Epidemiological data have implicated dietary fat as an important factor in the aetiology of various cancers in humans. This suggestion is supported by the results of experiments with animals which have shown that increased amounts of dietary fat, in particular polyunsaturated fat, increase the incidence of some spontaneous and induced tumours. The enhancement of carcinogenesis by dietary fats appears to be exerted at the promotional stage of carcinogenesis. Changes induced by dietary fats in several biological systems involved in carcinogenesis may well indicate the underlying mechanisms responsible for the results of these experiments. These include the effects of dietary fats on the metabolism of chemical carcinogens, the structure and function of membranes, immunocompetence, DNA repair potential and endocrine function.

Action of histone on neoplastic and normal growth.

Histone, arginine-rich type IV, from calf thymus was tested for its effects on the healing of wounds in rabbit ears, on the regeneration of amputated limbs in the newt, on initiation and promotion stages of carcinogenesis of mouse skin and on incidence of sarcoma on films implanted in the mouse.