Peeyush K. Lala

Role of nitric oxide in carcinogenesis and tumour progression

Nitric oxide (NO) is a short-lived molecule required for many physiological functions, produced from L-arginine by NO synthases (NOS). It is a free radical, producing many reactive intermediates that account for its bioactivity. Sustained induction of the inducible form of NOS (iNOS) in chronic inflammation may be mutagenic, through NO-mediated DNA damage or hindrance to DNA repair, and thus potentially carcinogenic. Expression of iNOS is positively associated with P53 mutation in tumours of the colon, lung, and oropharynx. Progression of a large majority of human and experimental tumours seems to be stimulated by NO resulting from activation of iNOS or constitutive NOS, whereas inhibition is documented in others. This discrepancy is largely explained by differential sensitivity of tumour cells to NO-mediated cytostasis or apoptosis and clonal evolution of NO-resistant and NO-dependent cells. P53 mutation or loss is one of many events linked with NO resistance and dependence. NO can stimulate tumour growth and metastasis by promoting migratory, invasive, and angiogenic abilities of tumour cells, which may also be triggered by activation of cyclo-oxygenase (COX)-2. Thus, selective inhibitors of NOS, COX, or both may have a therapeutic role in certain cancers.

Role of nitric oxide in tumor progression : Lessons from experimental tumors

Nitric oxide (NO), a potent biological mediator, plays a key role in physiological as well as pathological processes, including inflammation and cancer. The role of NO in tumor biology remains incompletely understood. While a few reports indicate that the presence of NO in tumor cells or their microenvironment is detrimental to tumor cell survival and consequently their metastatic ability, a large body of clinical and experimental data suggest a promoting role of NO in tumor progression and metastasis. We suggest that tumor cells capable of very high levels of NO production die in vivo, and those producing or exposed to lower levels of NO, or capable of resisting NO-mediated injury undergo a clonal selection because of their survival advantage; they also utilize certain NO-mediated mechanisms for promotion of growth, invasion and metastasis. The possible mechanism(s) are: (a) a stimulatory effect on tumor cell invasiveness, (b) a promotion of tumor angiogenesis and blood flow in the tumor neovasculature, and (c) a suppression of host anti-tumor defense. In this review, we discuss these mechanisms on the basis of data derived from experimental models, in particular, a mouse mammary tumor model in which the expression of eNOS by tumor cells is positively correlated with invasive and metastatic abilities. Tumor-derived NO was shown to promote tumor cell invasiveness and angiogenesis. The invasion-stimulating effects of NO were due to an upregulation of matrix metalloproteases and a downregulation of their natural inhibitors. Treatment of tumor-bearing mice with NO-blocking agents reduced the growth and vascularity of primary tumors and their spontaneous metastases. We propose that selected NO-blocking drugs may be useful in treating certain human cancers either as single agents or as a part of combination therapies.

Mechanisms of trophoblast invasiveness and their control: the role of proteases and protease inhibitors

SummaryImplantation and subsequent placental development in many species including the human are dependent on trophoblast invasion of the uterine epithelium, the underlying basement membrane, connective tissue and blood vessels. However, trophoblast invasion in situ is strictly controlled by the microenvironment provided by the pregnant uterus.Key mechanisms underlying various steps in trophoblast invasion of basement membrane and stroma are similar to those identified in the case of invasive tumor cells: (a) attachment to basement membrane by binding to laminin and possibly other basement membrane components; (b) detachment from the basement membrane matrix prior to its penetration, a process that requires the presence of complex-type oligosaccharides on the cell surface; (c) breakdown of basement membrane components by tropholast-derived metalloproteases (type IV and interstitial collagenase) and serine proteases (plasminogen activator). Type IV collagenase activity is stimulated by binding to laminin, a molecule also secreted by the trophoblast. Activation of trophoblast-derived metalloproteases appears to be plasmin-dependent. Plasmin results from the cleavage of plasminogen by trophoblast-derived plasminogen activator.Control of trophoblast invasion in situ is mediated by decidua-derived transforming growth factor ß (TGFβ) which in turn induces tissue inhibitor of metalloproteases (TIMP) both in the decidua and the trophoblast. We suggest that this control of trophoblast invasiveness is regulated both spatially as well as temporally during gestation. A preprogrammed decline in trophoblast invasiveness with increasing gestational age remains an additional possibility. The nature of the loss of control of trophoblast invasiveness in choriocarcinoma remains to be identified. Refractoriness to TGFß action remains to strong possibility.

Cyclooxygenase inhibitors retard murine mammary tumor progression by reducing tumor cell migration, invasiveness and angiogenesis

Tumor‐derived prostaglandins (PGs) have been implicated in the progression of murine and human breast cancer. Chronic treatment with a non‐selective PG inhibitor indomethacin was shown in this laboratory to retard the development and metastasis of spontaneous mammary tumors in C3H/HeJ female retired breeder mice. The present study examined the role of endogenous prostaglandins in the proliferation/survival, the migratory and invasive behavior and angiogenic ability of a highly metastatic murine mammary tumor cell line, C3L5, originally derived from a C3H/HeJ spontaneous mammary tumor. This cell line was shown to express high levels of cyclooxygenase (COX) –2 mRNA and protein as detected by Northern and Western blotting as well as immunostaining. PGE2 production by C3L5 cells was primarily owing to COX‐2, since this was blocked similarly with non‐selective COX inhibitor indomethacin and selective COX‐2 inhibitor NS‐398, but unaffected with the selective COX‐1 inhibitor valeryl salicylate. C3L5 cell proliferation/survival in vitro was not influenced by PGs, since their cellularity remained unaffected in the presence of PGE2 or NS‐398 or PG‐receptor (EP1/EP2) antagonist AH6809; a marginal decline was noted only at high doses of indomethacin, which was not abrogated by addition of exogenous PGE2. Migratory and invasive abilities of C3L5 cells, as quantitated with in vitro transwell migration/invasion assays, were inhibited with indomethacin or NS‐398 or AH6809 in a dose‐dependent manner; the indomethacin and NS‐398‐mediated inhibition was partially reversed upon addition of exogenous PGE2. An in vivo angiogenesis assay that used subcutaneous implants of growth factor‐reduced matrigel inclusive of tumor cells showed a significant inhibition of blood vessel formation in these implants in animals treated with indomethacin compared with animals receiving vehicle alone. These studies show that selective and nonselective COX‐2 inhibitors retarded tumor progression in this COX‐2‐expressing murine mammary tumor model by inhibiting tumor cell migration, invasiveness and tumor‐induced angiogenesis. The inhibitory effects were not entirely PG dependent; some PG‐independent effects were also noted.

Characteristic's of trophoblast cells migrating from first trimester chorionic villus explants and propagated in culture

We developed a method of propagating pure first trimester human trophoblast cells growing out of primary explants of mechanically derived chorionic villus fragments (Yagel et al, 1989; Graham et al, 1992). We have now extensively characterized these cells during their initial outgrowth and in long-term culture, employing a variety of markers and techniques as outlined below. By double label immunofluorescence using epithelial (cytokeratin) and mesenchymal (vimentin) cell markers, we identified the chorionic villus migrant cell populations as pure trophoblast (39 per cent of outgrowths) or a mixture of trophoblast and fibroblast (61 per cent). Further phenotyping of the pure trophoblast outgrowths by double label immunostaining using anti-cytokeratin antibody and a panel of other primary antisera revealed that these cells exhibit a variety of markers characteristic of extravillous invasive trophoblast cells in situ: insulin-like growth factor (IGF)-II, NDOG-5, proliferating cell nuclear antigen (PCNA), human leucocyte antigen framework antigen (W6/32) and a distinct set of integrins including alpha 1, alpha 3, alpha 5, alpha v and beta 1 subunits and alpha v beta 3/beta 5 vitonectin receptor. They were negative for alpha 6 and beta 4 integrin subunits. Immunogold electron microscopy of explants grown on type IV collagen gel revealed the production of conventional and oncofetal types of fibronectin by mononucleate trophoblast cells and human placental lactogen by multinucleate cells. Immunolabelling, flow cytometry and immunoprecipitation revealed that this phenotypic profile was retained with complete fidelity in the long-term culture; thus, trophoblasts migrating out of first trimester chorionic villus explants and their propagated progeny belong to the invasive extravillous trophoblast of the placenta.

Nitric Oxide Synthase Inhibition by NG-Nitro-l-Arginine Methyl Ester Inhibits Tumor-Induced Angiogenesis in Mammary Tumors

Using a murine breast cancer model, we earlier found a positive correlation between the expression of nitric oxide synthase (NOS) and tumor progression; treatment with inhibitors of NOS, N(G)-methyl-L-arginine (NMMA) and N(G)-nitro-L-arginine methyl ester (L-NAME), had antitumor and antimetastatic effects that were partly attributed to reduced tumor cell invasiveness. In the present study, we used a novel in vivo model of tumor angiogenesis using subcutaneous implants of tumor cells suspended in growth factor-reduced Matrigel to examine the angiogenic role of NO in a highly metastatic murine mammary adenocarcinoma cell line. This cell line, C3L5, expresses endothelial (e) NOS in vitro and in vivo, and inducible (i) NOS in vitro on stimulation with lipopolysaccharide and interferon-gamma. Female C3H/HeJ mice received subcutaneous implants of growth factor-reduced Matrigel inclusive of C3L5 cells on one side, and on the contralateral side, Matrigel alone; L-NAME and D-NAME (inactive enantiomer) were subsequently administered for 14 days using osmotic minipumps. Immediately after sacrifice, implants were removed and processed for immunolocalization of eNOS and iNOS proteins, and measurement of angiogenesis. Neovascularization was quantified in sections stained with Massons trichrome or immunostained for the endothelial cell specific CD31 antigen. While most tumor cells and endothelial cells expressed immunoreactive eNOS protein, iNOS was localized in endothelial cells and some macrophages within the tumor-inclusive implants. Measurable angiogenesis occurred only in implants containing tumor cells. Irrespective of the method of quantification used, tumor-induced neovascularization was significantly reduced in L-NAME-treated mice relative to those treated with D-NAME. The quantity of stromal tissue was lower, but the quantity of necrotic tissue higher in L-NAME relative to D-NAME-treated animals. The total mass of viable tissue (ie, stroma and tumor cells) was lower in L-NAME relative to D-NAME-treated animals. These data suggest that NO is a key mediator of C3L5 tumor-induced angiogenesis, and that the antitumor effects of L-NAME are partly mediated by reduced tumor angiogenesis.

Nitric oxide promotes murine mammary tumour growth and metastasis by stimulating tumour cell migration, invasiveness and angiogenesis

The contributory role of nitric oxide (NO) on tumour growth and metastasis was evaluated in a murine mammary tumour model. NO synthase (NOS) protein expression levels were examined in spontaneously arising C3H/HeJ mammary adenocarcinomas and respective lung metastases. In addition, 2 clonal derivatives of a single spontaneous tumour differing in metastatic phenotype (C3L5 and C10; highly and weakly metastatic, respectively) were utilised to investigate (i) the relationship between NOS expression levels and the biological behaviour of tumour cells (e.g., in vitro migratory and invasive capacities, in vivo tumour growth rate and metastatic and angiogenic capacities) and (ii) whether tumour‐derived NO stimulated the invasive, migratory and angiogenic capacities of tumour cells. A heterogeneous pattern of endothelial NOS (eNOS) expression was observed in tumour cells in spontaneous primary tumours, and eNOS expression was higher in undifferentiated relative to differentiated tumour zones. However, tumour cells in lung metastatic sites were always strongly eNOS‐positive, suggesting that eNOS expression facilitated metastasis. Findings using clonal derivatives supported this notion; s.c. primary tumour growth rate, efficiency of spontaneous metastasis and eNOS expression were higher for C3L5 relative to C10 cell lines. Nevertheless, lung metastases derived from both tumour cell lines were always strongly and homogeneously eNOS‐positive. C3L5 cells were more invasive than C10 cells in vitro, but the migratory capacities of the cell lines did not differ. However, migration and invasiveness of both cell lines were inhibited with L‐NAME and restored with excess L‐arginine. Tumour‐associated angiogenesis, measured in Matrigel implants inclusive of tumour cells, was higher for C3L5 relative to C10 cells, and C3L5‐induced angiogenesis was reduced with chronic L‐NAME treatment of host animals. These findings suggest that tumour‐derived eNOS promoted tumour growth and metastasis by multiple mechanisms: stimulation of tumour cell migration, invasiveness and angiogenesis. Int. J. Cancer 86:30–39, 2000.

Factors Regulating Trophoblast Migration and Invasiveness: Possible Derangements Contributing to Pre-eclampsia and Fetal Injury

Impaired trophoblast invasiveness and spiral arterial remodelling, which results in poor placental perfusion during early pregnancy, is believed to cause fetal injury and growth retardation, and also endothelial cell activation/dysfunction in a susceptible mother, leading to clinical manifestations of pre-eclampsia. This article briefly reviews the regulatory roles of certain locally active factors in trophoblast migration and invasiveness. This background is then used to discuss and debate whether derangements or dysfunction of some of these factors can manifest as early serum markers predictive of the disease, as opposed to the intermediate and late stage markers which may reflect manifestations and consequences of the disease. Of particular significance are the observed derangements in uPA/uPAR/PAI system, IGFBP-1, HGF, HB-EGF and TGFbeta, factors which are known to regulate trophoblast migration and invasiveness in situ. An emphasis is placed on the need for longitudinal studies in order to identify predictive serum markers which may help strategies for prevention or amelioration of fetal injury and pre-eclampsia.

Role of prostaglandin E2 receptors in migration of murine and human breast cancer cells

Aberrant upregulation of COX-2 enzyme resulting in accumulation of PGE2 in a cancer cell environment is a marker for progression of many cancers, including breast cancer. Four subtypes of cell surface receptors (EP1, EP2, EP3, and EP4), which are coupled with different G-proteins, mediate PGE2 actions. Since migration is an essential step in invasion and metastasis, in the present study we defined the expression of EP receptors and their roles in migratory function of breast cancer cells of murine (C3L5) and human (MDA-MB-231 and MCF-7) origin. Highly metastatic C3L5 and MDA-MB-231 cells, found to be highly migratory in a Transwell migration assay, were shown to accumulate much higher levels of PGE2 in culture media in comparison with nonmetastatic and poorly migrating MCF-7 cells; the levels of PGF2alpha and 6-keto-PGF1alpha were low in all cases. The elevated PGE2 production by metastatic cancer cells was due to COX-2 activity since dual COX-1/2 inhibitor indomethacin and selective COX-2 inhibitor NS-398 equally suppressed both basal and inducible (by IFN-gamma/LPS or Ca2+-ionophores) PGE2 accumulation. RT-PCR analysis revealed that murine C3L5 cells expressed mRNA of EP1, EP3, and EP4 but not EP2 receptors. On the other hand, human MDA-MB-231 and MCF-7 cells expressed all the above receptors. High levels of expression of functional EP4 receptors coupled with Gs-protein was confirmed in C3L5 cells by biochemical assay showing a dose-dependent increase of intracellular cAMP synthesis in response to PGE2. EP receptor antagonists SC-19220, AH-6809, and AH-23848B, having highest affinity for EP1, EP1/EP2/DP, and EP4 receptors, respectively, variably inhibited migration of metastatic breast cancer cells. An autocrine PGE2-mediated migratory activity of these cells appeared to be associated predominantly with EP4 receptor-mediated signaling pathway, which uses cAMP as a second messenger. This conclusion is based on several observations: (1) selective EP4 antagonist AH-23848B effectively inhibited migration of both C3L5 and MDA-MB-231 cells in a dose-dependent manner; (2) exogenous PGE2 and EP4 agonist PGE1 alcohol increased migration of C3L5 cells; (3) forskolin, a potent activator of adenylate cyclase, as well as membrane-permeable analogues of cAMP (8-bromo-cAMP, dibutyryl-cAMP) stimulated migration of C3L5 cells; and (4) Rp-cAMPS, a selective protein kinase A inhibitor, reduced migration of C3L5 cells. Migration of poorly migratory MCF-7 cells remained unaffected with either PGE2 or EP4 antagonist. These findings are relevant for designing therapeutic strategies against breast cancer metastasis.

PGE2-mediated immunosuppression by first trimester human decidual cells blocks activation of maternal leukocytes in the decidua with potential anti-trophoblast activity☆

We have earlier shown that first trimester human decidual cells and decidual macrophages suppress T lymphocyte alloreactivity in an MHC-unrestricted manner by secreting PGE2, which blocks the generation of IL-2 receptors (IL-2R) and production of IL-2 by lymphocytes but does not interfere with the interaction between IL-2 and IL-2R or the lytic function of CTL, once generated. The present study examined whether these events constituted a physiological, immunoprotective mechanism in situ against the activation of maternal decidua-infiltrating leukocytes with potential anti-trophoblast cytocidal function. We examined (1) whether there was IL-2R expression, IL-2 production, or anti-trophoblast killer activity in short-term (0-3 day) cultures of collagenase-dispersed first trimester human decidua inclusive of leukocytes; (2) if not, whether any of these parameters could be stimulated in these cultures by blocking PGE2 synthesis with indomethacin, or neutralizing PGE2 with anti-PGE2 antibody; (3) whether exogenously added recombinant IL-2 in the presence or absence of indomethacin stimulated IL-2R expression or anti-trophoblast killer function in these cultures. IL-2R (as defined by Tac antigen) was measured in the whole cell population by a radioimmunoassay and further examined at the cellular level with radioautography. IL-2 production in culture supernatants was measured from the proliferative response (3HTdR uptake) of an IL-2-dependent (CTLL) cell line. Killer activity in fresh or cultured decidua-associated cells as well as PBL of normal or pregnant subjects was measured against 51Cr-labeled targets inclusive of autologous cytotrophoblast cells or long-term human trophoblast cell lines, K562 and Daudi cells. Results revealed a complete absence of IL-2R expression, IL-2 production, or anti-trophoblast killer activity in the untreated cultures of the decidua, but all these parameters were significantly stimulated in the presence of indomethacin or anti-PGE2 antibody. The indomethacin-stimulated killer cells had NK-like activity. Presence of high dose exogenous IL-2 alone in these cultures strongly stimulated IL-2R expression and anti-trophoblast killer function, which were augmented further in the additional presence of indomethacin. The resultant killer cells had LAK cell-like activity. These findings suggest that PGE2 secretion by first trimester human decidual cells blocks activation of maternal leukocytes in the decidua with potential anti-trophoblast killer function, by inhibiting IL-2 receptor generation and IL-2 production in situ.