Antiproliferative Activity via Signal Transduction Pathways

Much is now known about the biochemical control mechanisms involved in regulating cell growth and development. Cells respond to signals from extracellular stimuli via a complicated network of highly regulated events collectively referred to as signal transduction pathways. Stimulation of these pathways results in changes in transcriptional activity. While normal cells respond appropriately to extracellular stimuli, many precancerous and cancerous cells have lost this ability and display aberrant signalling.

Molecular targets on these pathways that allow interference with the deregulated signalling are potential sites for chemopreventive intervention (Powis, 1994; Powis and Workman, 1994). For example, key components of these pathways are growth factors such as epidermal growth factor (EGF), insulin-like growth factor (IGF) and transforming growth factor (TGF) and protein tyrosine kinases which catalyse the transfer of the phosphate of ATP to the hydroxyl group of tyrosine on numerous proteins. Many growth factor receptors (e.g. EGFR) and oncogenes are tyrosine kinases, and loss of tyrosine kinase regulatory mechanisms has been implicated in neoplastic growth.

Invocation of signal transduction pathways also provides a mechanistic rationale for the multiple chemopre-ventive effects of some classes of agents. For example, chemopreventive agents such as retinoids, antihormones and protein kinase inhibitors which affect activities at the cell membrane level and cytoplasmic and nuclear receptor levels can also affect other connected events. It is evident that many of these activities are interrelated, e.g. effects on the proliferation associated enzyme ornithine decarboxylase (ODC), AA metabolism, protein kinase C (PKC), IGF-I and TGF may be pleiotropic results of activity at single locus on signal transduction pathways. It is also clear that a single activity may not be the most important or the only one required for carcinogenesis. (See section on The Molecular Basis of Cell and Tissue Organisation.)

Modulate Hormonal/Growth Factor Activity

Chemicals may inhibit the proliferation associated with carcinogenesis by directly regulating the induction and activity of specific hormones and growth factors that initiate steps in signal transduction (Kelloff et al, 1995b). This regulation may occur at membrane level receptors (for growth factors, peptide hormones and neurotransmitters) or via cytoplasmic and nuclear receptors (for the steroid superfamily consisting of oestrogen, progesterone, retinoid, glucocorticoid, vitamin D and thyroid receptors). For example, antioestrogens such as tamoxifen bind to nuclear oestrogen receptors, preventing the binding and activity of oestrogens. Tamoxifen inhibits carcinogen-induced, oestrogen-sensitive tumours in rat mammary glands and hamster kidney. Most importantly, tamoxifen has been shown to lower the risk of breast cancer in women at high risk. Phyto-oestrogens, such as the isoflavone genistein, have anti-oestrogenic activity. Studies in human breast cancer cells indicate that the anti-oestrogenic effect may result from slowed translocation ofgenistein-bound receptor from the cytoplasm to the nucleus compared with that of oestradiol-bound receptor.

TGF/3 has antiproliferative activity in both normal and cancer cells. These observations suggest that chemicals that activate TGF3 could also control proliferation in car-cinogenesis. In this regard, breast cancer cells normally produce only small amounts of activated TGF3, but treatment with tamoxifen increases production up to 20-fold. Retinoic acid, which inhibits chemical carcinogenesis, particularly tumour promotion in mouse skin, induces TGF/32 in mouse skin after topical application. In vitamin A-deficient rats treated with retinoic acid, the level of expression of TGFcorrelates with levels of retinoids in skin, intestine and respiratory tract tissue.

There is also evidence of cross-regulation among membrane and nuclear receptors. For example, IGF-I stimulates cell replication in various tumours. Particularly, human breast cancer cells have membrane receptors for and excrete IGF-I. Tamoxifen lowers blood concentrations of IGF-I in breast cancer patients, suggesting that part of its antitumour activity is inhibition of IGF-I.

Other aspects of receptor activity are possible mechanisms for chemopreventive activity. Generally, receptors are phosphoproteins, and phosphorylation appears to play a role in receptor activation. Thus, chemopreventive agents which inhibit phosphorylation, e.g. inhibit protein kinases, may influence cell proliferation by effects on receptors. An example is the isoflavone genistein, which is a specific inhibitor of tyrosine kinase and other flavonoids.

Deactivation of steroids may prevent hormone-stimulated carcinogenesis. In this regard, aromatase inhibitors and modifiers of steroid hydroxylation have been described above under inhibition of carcinogen formation/activation and carcinogen deactivation/detoxification, respectively.

Inhibit Oncogene Activity

During the course of cell proliferation in carcinogenesis, numerous oncogenes are expressed abnormally, possibly as intermediates in the signal transduction pathways. The evidence for oncogene activity in signal transduction pathways is based on the similarity of some of their products (protein kinases) to other intermediates in these pathways (Kelloff et al., 1995b). There are several points during activation at which the ras oncogene can be inhibited, and there are data relating this inhibition to chemopreventive activity.

First, a membrane receptor-linked tyrosine kinase is involved in Ras activation, and kinase inhibitors would be expected to prevent Ras activation. Particularly interesting are compounds such as genistein which specifically inhibit tyrosine kinase, and thus do not interfere with normal cellular processes mediated by other kinases.

To be activated, the Ras protein must first be farne-sylated. Ras oncogenes are involved in rat mammary gland carcinogenesis. D-Limonene inhibits the progression of carcinogen-induced mammary tumours induced in rats, and it also inhibits the farnesylation of small G proteins (21-26 kDa); these experimental data suggest that D-limonene could be preventing oncogene activation by inhibiting post-translational farnesylation of the p21 Ras protein. Perillyl alcohol is an even more potent inhibitor of farnesyl--protein transferase. Recently, several specific farnesylation inhibitors have been described which are structural analogues of the C-terminal tetrapeptide of farnesyl--protein transferase and inhibit the growth of ras-dependent tumours.

Further, farnesyl pyrophosphate, the substrate for farnesyl--protein transferase, is an intermediate in the synthetic pathway from hydroxymethylglutaryl coenzyme A (HMG CoA) reductase to cholesterol. Inhibitors of HMG CoA reductase, e.g. lovastatin, and probably inhibitors of other enzymes along the synthetic route to cholesterol, have been shown to inhibit Ras farnesylation.

Cyclooxygenase (COX) catalyses the synthesis of prostaglandins (PGs) from AA. COX inhibitors also might inhibit proliferation in carcinogenesis by inhibition of oncogene expression, although the evidence is less direct than for other effects of AA metabolism inhibitors. Expression of the oncogene c-myc occurs early in EGF-induced cell proliferation. PGs are required but not sufficient for c-myc expression and DNA synthesis stimulated by EGF. The NSAID indomethacin inhibits both EGF-induced DNA synthesis and oncogene expression; this inhibition is reversed by addition of PGG2.

Studies in vitro indicate inhibition of oncogene expression as a mechanism for chemopreventive activity of protease inhibitors and retinoids. For example, the protease inhibitors inhibit transformation of cells trans-fected with activated H-ras oncogene, suppress c-myc expression in mouse fibroblasts and inhibit carcinogen-induced tumours in rat colon, mouse lung and mouse skin. Retinoic acid also inhibits H-ras-induced transformation in cancer cells and mouse skin carcinogenesis.

Inhibit Polyamine Metabolism

Polyamines play a significant role in cell proliferation, differentiation and malignant transformation. The mode of action is not yet known, but it has been suggested that polyamines stabilize DNA structures; they have been shown to affect DNA and protein synthesis. A critical step in polyamine biosynthesis is the synthesis of putrescine from ornithine that is catalysed by ODC. There is ample evidence that ODC participates in carcinogenesis -- the enzyme is induced during cell transformation by chemical carcinogens, viruses and oncogenes.

Association with cell proliferation during carcinogen-esis is also well established. TPA and other tumour promoters increase ODC activity in skin, colon, bladder and liver. In mouse skin, topically applied TPA causes an approximately 200-fold increase in ODC activity within 4.5 h after treatment. The increase is dose dependent and correlates with the ability of the TPA dose to promote skin tumours. Also, the increased ODC activity has been proposed to be specific to tumour promotion, since most carcinogens that are not tumour promoters do not induce ODC.

Likewise, chemicals that inhibit induction of or deactivate ODC also inhibit carcinogenesis. Some of the most convincing results demonstrating that inhibition of ODC prevents cancers come from studies with DFMO. DFMO is a specific, mechanism-based irreversible inhibitor of ODC - that is, DFMO is activated by ODC into a form that reacts with the enzyme to inactivate it. DFMO inhibits carcinogen-induced tumours in mouse and rat colon and bladder, rat mammary glands and mouse skin.

ODC induction by TPA is regulated at the transcription level. Regulation occurs in part via signal trans-duction events at the membrane. For example, PKC appears to be involved, as are diverse signal transduction intermediates induced by TPA, including PGs, other products of AA metabolism and free radicals. Chemicals that inhibit PKC and AA metabolism and those that scavenge free radicals also may inhibit the induction of ODC, hence they may be chemopreventives by this mechanism. In this regard, several of the PKC inhibitors, including glycyrrhetinic acid, inhibit ODC induction and tumour promotion in mouse skin. AA metabolism inhibitors also inhibit both ODC induction and TPA-promoted mouse skin tumorigenesis, as do free radical scavengers such as GSH, flavonoids and green tea polyphenols.

Vitamin A (retinol) and its derivatives (i.e. retinoids) inhibit carcinogenesis specifically during promotion. There is evidence that the cancer inhibitory activity of these compounds may be mediated partially by regulation of ODC induction. One of the most active retinoids is fenretinide. This compound is a potent inhibitor of ODC induction as well as TPA promotion in mouse skin. It also inhibits carcinogen-induced mammary gland tumours in rats and bladder tumours in mice.

Inhibition of S-adenosyl-L-methionine (SAM) decarboxylase is another mechanism for inhibiting polyamine biosynthesis that may prove useful in chemoprevention. This enzyme, like ODC, is highly regulated in mammalian cells and catalyses the formation of the polyamines sper-midine and spermine from putrescine.

Induce Terminal Differentiation

Terminal differentiation is one of the steps in normal, regulated cell proliferation in epithelial tissues. Proliferating cancer cells often have lost the ability to differentiate. These cancer cells are either deficient in or incapable of responding to differentiation signals. Abundant evidence demonstrates that restoring the ability of abnormally proliferating cells to differentiate suppresses carcinogenesis. Several classes of chemopreventives also induce differentiation. Retinoids are the best-studied example (Singh and Lippman, 1998a). For many years it has been known that vitamin A deficiency causes squamous metaplasia and hyperkeratinization -- both are signs of excessive tissue. Studies in hamster trachea and various cancer cells show that the differentiated phenotype can be restored by treatment with retinoids. Evidence indicates that retinoids control differentiation via intracellular binding proteins (cellular retinol-binding protein and cellular retinoic acid-binding protein) and nuclear receptors.

Calcium and vitamin D3 are well-known differentiating agents that also inhibit carcinogenesis. Calcium induces differentiation in epithelial tissues including rat oesophagus, mouse skin and human mammary gland and colon. Vitamin D3 induces differentiation in human colon, human and mouse myeloid leukaemia cells, mouse skin cells, mouse melanoma cells and other cells. It has been suggested that the effects of the two chemicals on differentiation may be mediated by the same signal transduction pathway, involving the vitamin D3 nuclear receptor with calcium as the messenger.

Restore Immune Response

Antibodies to oncogene products are important in the inhibition of cell transformation and tumour growth. PGE2 is known to suppress immune response in certain tumour cells. COX inhibitors diminish the immune suppression, and it has been suggested that this effect on immune suppression may be part of the mechanism by which COX inhibitors reduce tumour growth, as seen in several animal tumour models including colon and Lewis lung carcinoma.

Retinoids also are known to be immunostimulants. Retinoic acid increases cell-mediated and natural killer (NK) cell cytotoxicity; retinoids also cause some leukaemia cells to differentiate to mature granulocytes comparable to mature neutrophils. These effects might be partially responsible for the activity of retinoids against established tumours.

Pharmacological doses of vitamin E fed with normal, well-balanced animal diets increase humoural antibody production, especially IgG; this effect has been observed repeatedly in chickens, mice, turkeys, guinea pigs and rabbits. Vitamin E also stimulates cell-mediated immunity, as evidenced by enhanced mitogenesis and mixed lymphocyte response in spleen cells from mice fed the vitamin. In particular, vitamin E prevents the carcinogen-induced decrease in the density of macrophage-equivalent cells (Langerhans cells) in the buccal pouch of carcinogen-treated hamsters. Likewise, vitamin E inhibits the induction and causes regression of tumours in hamster buccal pouch.

The role of selenium in mediating immune responses suggests that the broad spectrum activity of selenium in inhibiting chemical carcinogenesis may be attributed partially to stimulation of the immune system. In general, selenium deficiency causes immunosuppression, while supplementation with low doses of selenium restores and increases immune response. Perhaps most important in inhibiting tumorigenesis is the effect of selenium on the cytotoxicity of immune system cells. Compared with normal cells, both T and NK lymphocytes from selenium-deficient mice have decreased ability to destroy tumour cells in vitro. Supplementation with selenium enhances the ability of rat NK cells to kill tumour cells. The role of immunostimulation in carcinogenesis inhibition by selenium has been studied to only a limited extent and has not been confirmed. However, the potent inhibitory activity of selenium compounds against DMBA-induced tumors in rat mammary glands is suggestive, since the immunosup-pressive effects of DMBA are well documented.

Increase Intercellular Communication

Gap junctions are the cell components that coordinate intercellular communication. They are composed of pores, or channels, in the cellular membranes that join channels of adjacent cells; these pores are regulated and, when open, allow passage of molecules up to about 1000 D in size. Gap junctions may allow growth regulatory signals to move between cells. There is evidence from studies in vitro that inhibition of gap junctional intercellular communication occurs in the proliferative phase of carcinogenesis. In in vitro studies, enhancement of communication correlates to inhibition of cellular transformation.

To date, only limited data suggest the potential for inhibiting chemical carcinogenesis by the other antipro-liferative/antiprogression mechanisms listed in Table 1, but the possibilities exist and warrant consideration here.

Restore Tumour-suppressor Function

Many so-called 'tumour-suppressor' genes have been found that may be involved in controlling proliferation and differentiation in cells. Particularly, their function is associated with control of abnormal growth in carcinogenesis. Several of these genes have been identified and implicated in pathogenesis by the presence of mutated or otherwise dysfunctional forms in specific cancers. For example, the tumour suppressor Rb is involved in retinoblastoma, osteosarcoma and tumours in lung, bladder, prostate and breast; p53 in adenocarcinomas in colon and breast, human T cell leukaemias, glioblastomas, sarcomas, and tumours in lung and liver; WT in Wilm tumour; and DCC (Deleted in Colorectal Cancer) in colon tumours. There is potential for treating cancer patients with exogenous functional tumour-suppressor genes to inhibit tumour growth and spread. Possibly, it also will be found that chemicals can modulate the expression and activity of tumour suppressors and inhibit carcinogenesis by this mechanism. CP-31398 stabilizes the DNA binding domain of both normal and mutant p53 in an active conformation, induces the p21WAFI cell cycle regulatory protein in the absence of normal p53 and inhibits growth of human tumours with p53 mutated tumours in a mouse model.

Induce Programmed Cell Death (Apoptosis)

Apoptosis is a well-regulated function of the normal cell cycle requiring gene transcription and translation. Tumour suppressors, such as p53 and certain regulatory growth factors, particularly TGF/31, have been implicated as inducers of apoptosis. Programmed cell death has been described as the complement to mitosis in the maintenance, growth and involution of tissues; it is the process by which damaged and excessive cells are eliminated. Apoptosis is inhibited by tumour promoters such as TPA and phenobarbital and other chemicals that stimulate cell proliferation such as hormones. These data suggest that induction of apoptosis may inhibit tumour formation and that agents which inhibit tumour promotion may act by inducing or preventing inhibition of apoptosis through any one of several signal transduction pathways. For example, hamster pancreatic cancers regress when apoptosis is induced, and many potential chemopreventive agents (e.g. tamoxifen, NSAIDs, retinoids) induce programmed cell death in precancerous and cancer cells.

Correct DNA Methylation Imbalances

Changes in DNA methylation patterns appear to be involved in carcinogenesis. (See the chapter on Non-Genotoxic Causes of Cancer.) Methyl-deficient diets cause fatty livers, increased cell turnover and promote the development of carcinogen-induced liver tumours in rats and mice. Conversely, methyl-rich (fortified with choline and methionine) diets prevent or reduce these effects. Changes in gene expression, such as increased expression of oncogenes, appear in animals on methyl-deficient diets. These effects are similar to those seen in rodents given tumour-promoting chemicals and they are reversible on methyl replacement. Hypomethylation is also associated with hyperproliferation in colon tissue. Methionine, which is involved with choline, folic acid and vitamin B12 in regulating intracellular methyl metabolism inhibits carcinogen-induced mammary gland cancers in rats. Also, folic acid inhibits carcinogen-induced lung tumours in mice. Conversely, methylation of CpG islands in the promoter regions of tumour-suppressor and GST genes has been seen in cancers of several major target organs including colon, prostate, breast and lung. This methylation prevents gene expression and provides a rationale for the chemopreventive activity of agents which induce GST and tumour-suppressor activity.

Inhibit Angiogenesis

Angiogenesis is the process leading to the formation of new blood vessels. In normal tissue, it is a highly regulated process essential to reproduction, development and wound repair. In carcinogenesis, it is required in tumour growth and involved in metastasis, and there is evidence that angiogenesis also may occur early in carcinogenesis. There is indirect evidence that certain chemicals that inhibit carcinogenesis may inhibit angiogenesis. For example, PGs E1 and E2 are angiogenic. Therefore, agents that inhibit PG synthesis may inhibit carcinogenesis by inhibiting angiogenesis. Similarly various growth factors and, particularly, vascular endothelial growth factor (VEGF), increase angiogenesis by activating signal transduction pathways. Inhibition of angiogenesis may be a chemopreventive mechanism for agents which affect these pathways.

Inhibit Basement Membrane Degradation

Tumour cells produce various enzymes that destroy the basement membrane which acts as a barrier against malignant cancer cells and prevents cancer spread. These enzymes include, among others, the proteases collagenase, cathepsin B, plasminogen activators and prostate-specific antigen (PSA). Protease inhibitors are known to act against thrombin and type IV collagenase, which are among the proteases hypothesized to participate in the destruction of basement membranes during cancer invasion. Proteases are also involved in angiogenesis. Thus, protease inhibitors that slow carcinogenesis may derive their effects, in part, by inhibiting basement membrane degradation or by inhibiting angiogenesis.

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