The alpha1 subunit of the sodium pump could represent a novel target to combat non-small cell lung cancers.

With an overall 5 year survival rate as low as 15% for non-small cell lung cancer (NSCLC), even with surgical intervention and the use of newer molecules in adjuvant chemotherapy, there is an urgent need for new biological targets and associated novel anti-cancer agents. The present study was undertaken to evaluate the potential of the Na(+)/K(+)-ATPase alpha1 subunit as a novel target in NSCLC and revealed that alpha1 expression is markedly higher in a significant proportion of NSCLC clinical samples compared to normal lung tissue. Furthermore, reduction in alpha1 expression in A549 NSCLC cells by anti-alpha1 siRNA resulted in markedly impaired proliferation and migration of these cancer cells. Finally, of three cardenolides investigated, UNBS1450, which is known to bind to Na(+)/K(+)-ATPase and displays potent anti-tumour activity in vivo in experimental models of human NSCLCs, is the most potent inhibitor of Na(+)/K(+)-ATPase isozymes (alpha1beta1, alpha2beta1 and alpha3beta1), most strikingly of alpha1beta1. This was reflected in the compound's more potent anti-proliferative activity in all NSCLC cell lines evaluated (A549, Cal-12T, NCI-H727 and A427); the first three of which over-express alpha1. The marked impairment in A549 NSCLC cell proliferation and migration, and resulting similar morphology following anti-alpha1 siRNA or UNBS1450 treatment, was associated with features of abnormal cytokinesis, mediated in the case of UNBS1450 by disorganization of the actin cytoskeleton. Collectively these data strongly suggest that targeting the Na(+)/K(+)-ATPase alpha1 using specific cardenolides could represent a novel means to combat certain NSCLCs.

New calamenene sesquiterpenes from Tarenna madagascariensis.

Four calamenene sequiterpenes, (+)-(7R,10S)-15-hydroxycalamenene (3), (+)-(7R,10S)-2,15-dihydroxycalamenene (4), (+)-(7R,10S)-2-hydroxy-15-calamenal (5), (+)-(7R,10S)-15-calamenal (6), along with the amorphane sesquiterpene (+)-(1S,6R,7R,10S)-1-hydroxy-3-oxo-amorph-4-ene (16), have been isolated from the Madagascan shrub Tarenna madagascariensis (Rubiaceae) and their structures determined by spectroscopic methods and chemical correlations. Furthermore, five known related sesquiterpenes [(+)-(7R,10S)-2-hydroxycalamenene (1), (+)-(7R,10S)-3-hydroxycalamenene (2), (-)-alpha-cadinol (13), cadinenal (14), 6-epicadinenal (15)], and three known lignans [(-)-hinokinin, (-)-dihydrocubebin, (-)-cubebin] were also isolated from the same plant. This is the first report of compounds 3, 4, 5, 6, and 16 from a natural source.

New terpenoids from two Indonesian marine sponges.

A C16 norsesterterpenoid (euplectellodiol, 1) and a norditerpenoid (2) have been isolated from the marine sponges Mycale euplectelloides and Diacarnus megaspinorhabdosa, respectively. Their structures have been determined by spectroscopic methods. Compounds 1 and 2 are new natural products.

Synthesis and cytotoxicity on sensitive and doxorubicin-resistant cell lines of new pyrrolo[2,1-c][1,4]benzodiazepines related to anthramycin.

A new 7,8-methylenedioxy analogue (4) of (+)-porothramycin B (2) and its water-soluble sodium bisulfite derivative (15) have been synthesized in high yields and have been shown to exhibit high cytotoxic activities against several tumor cell lines. The new pyrrolo[2,1-c][1,4]benzodiazepine 4 was as effective against the resistant cell lines as against the doxorubicin-sensitive cell lines tested.

Synthesis and characterization of the antitumor activities of analogues of meridine, a marine pyridoacridine alkaloid.

Marine compounds with pyridoacridine skeletons are known to exhibit interesting antitumor activities. Among these compounds, meridine has already been reported as having significant antitumor activities in vitro. We synthesized 24 analogues of meridine substituted on ring A with the aim of obtaining compounds that display significantly higher in vitro antitumor activities than meridine. The 24 compounds and meridine used as a control compound were tested at 6 different concentrations on 12 different human cancer cell lines including various histopathological types (glioblastomas and breast, colon, lung, prostate, and bladder cancers). The IC(50) value (i.e., the drug concentration inhibiting the mean growth value of the 12 cell lines by 50%) of these 25 compounds ranged over 5 log concentrations, i.e., between 10 and 0.0001 microM, with four of the compounds exhibiting a significantly higher in vitro antitumor activity than meridine. These compounds will now be subjected to further pharmacological investigation including in vivo testing on both conventional murine tumors and human tumors grafted onto nude mice.

The levels of retinoid RARbeta receptors correlate with galectin-1, -3 and -8 expression in human cholesteatomas.

Cholesteatoma is a benign disease characterized by the presence of an unrestrained growth and the accumulation of keratin debris in the middle ear cavity. This often recurs, even when surgical resection is thought to be complete. In a previous study we showed that cholesteatomas with the highest apoptotic indices recurred more rapidly and also exhibited a high level of p53 immunopositive cells. In view of their relevance to the characterization of the cell differentiation status, the present study focuses on the expression of retinoid acid receptors (RARs) and galectins in human cholesteatomas. Retinoids control the differentiation processes in keratinocytes while galectins play strikingly modulatory roles at apoptosis and cell adhesion levels in a wide variety of tissue (embryonic, normal and neoplastic). To clarify the expression of these two protein families in human cholesteatomas we examined and quantified the levels of immunohistochemical expression of RARalpha, beta and gamma, and also galectin-1, -3 and -8 in a series of 70 human cholesteatomas. Our data show clearly that predominantly RARbeta and galectin-1 were expressed. The RARgamma concentration was significantly lower than that of the RARalpha; this was also observed for the galectin-8 concentration in comparison with the galectin-3 one. Furthermore, the level of RARbeta expression correlated highly (P=0.00001) with the level of galectin-8 expression, which also correlated significantly with the level of RARalpha and RARgamma expression. In addition, this parameter also correlated with the level of galectin-1 and galectin-3 expression. These data suggest that cholesteatomas may originate in an undifferentiated population of keratinocytes, and that a relation may exist between retinoid activity and galectins.

Evidence for stimulation of tumor proliferation in cell lines and histotypic cultures by clinically relevant low doses of the galactoside-binding mistletoe lectin, a component of proprietary extracts.

The toxic galactoside-specific lectin from mistletoe, a component of proprietary extracts with unproven efficacy in oncology, exhibits capacity to trigger enhanced secretion of proinflammatory cytokines at low doses (ng/ml or ng/kg body weight) and reductions of cell viability with increasing concentrations. To infer any tumor selectivity of this activity, cytofluorimetric and cell growth assays with a variety of established human tumor cell lines were performed. Only quantitative changes were apparent, and the toxicity against tumor cells was within the range of that of the tested fibroblast preparations from 5 donors. No indication for any tumor selectivity was observed. In kinetic studies with 8 sarcoma and 4 melanoma lines, this evidence for quantitative variability of the response in interindividual comparison was further underscored. At 50 pg lectin/ml x 10(5) cells, even a growth-stimulatory impact was noted in 5 of 12 tested cases. To mimic in vivo conditions with presence of cytokine-secreting inflammatory and stromal cells, exposure to the lectin was extended to histotypic cultures established from 30 cases of surgically removed tumor. As salient result, 5 specimens from 4 of the 8 tested tumor classes responded with a significant increase of [3H]-thymidine incorporation relative to controls during the culture period of 72 hours, when the lectin was present at a concentration in the described immunomodulatory range (1 ng/ml). A relation of this activity to the extent of the actual proliferative status of the reactive samples could not be delineated. Therefore, a non-negligible percentage of the established tumor cell lines (e.g., 3 from 8 sarcoma lines) can be markedly stimulated by the lectin at a very low dose and with dependence on the cell type. Furthermore, the feasibility to elicit a significant growth enhancement is likewise documented for human tumor explants in 16.6% of the examined cases. In view of the uncontrolled application of lectin-containing extracts in alternative/complementary medicine, the presented results on unquestionably adverse lectin-dependent effects in two culture systems call for rigorous examination of the clinical safety of this unconventional, scientifically entirely experimental treatment modality.

Characterization of TNP-470-induced modifications to cell functions in HUVEC and cancer cells.

The aim of the present work is to characterize (both in vitro and in vivo) the influence of TNP-470 on different cell functions involved in angiogenesis and, more particularly, on endothelial cell growth, cell migration and vessel formation. In addition, a possible direct anti-tumor activity was investigated. To this end, we made use in vitro of human umbilical cord endothelial vein (HUVEC) cells and two human cancer cell lines. The TNP-470 effects on the growth of cancer cell lines were compared to those of Taxol (an inhibitor of microtubule depolymerization), a cytotoxic reference which also displays strong antiogenic activity at low (non-toxic) doses. The in vitro effects were characterized on the mouse mammary MXT adenocarcinoma, on which we also characterized the influence of three clinically active anti-tumor compounds (as cytotoxic references). The purpose of this part of the study was to determine the actual TNP-470-related anti-tumor activity and to evaluate the possible toxic side-effects at the doses at which this compound induces tumor growth inhibition. These investigations were completed by analyzing the TNP-470 effects on HUVEC cell motility and in vitro and in vivo vessel formation. The results show that in vitro, TNP-470 inhibited the growth not only of HUVEC, but also of neoplastic cells. Furthermore, TNP-470 clearly inhibited in vitro endothelial cell motility (p<10(-5)). However, it had only a minor effect (p=0.02) on the formation of HUVEC cell networks on Matrigel(R). In vivo, TNP-470 was able to inhibit tumor growth (on the MXT model) at a dose (50 mg/kg) associated with toxic side-effects. Histological examination showed a significant inhibition of vessel formation (p<0.001) at high (toxic) and intermediary (non-toxic) doses (50 and 20 mg/kg). However, we also observed that TNP-470 stimulated lymphocyte proliferation. Thus, care must be taken with the TNP-470 compound in combination with other anti-tumoral agents in order to avoid certain unfortunate clinical complications.

Characterization of steroid hormone sensitivity in human breast cancers maintained ex vivo under organotypical culture conditions.

PURPOSE: The methodology we propose combines the immunohistochemical determination of the oestrogen and progesterone receptors (ER and PgR) with the characterization of the oestradiol- and progesterone-induced influence on cell proliferation in breast cancers in order to characterize their steroid hormone sensitivity at both the "static" and "dynamic" level. METHODS: ER and PgR have been immunohistochemically quantified by means of computer-assisted microscopy. Cell proliferation has been determined by means of tritiated thymidine autoradiography in tumour samples maintained in vitro as organotypic cultures. A series of 14 patients was investigated. RESULTS: Of the 14 breast cancers under study, one with an unequivocally "very ER-rich"/"very PgR-rich" immunohistochemical phenotype totally failed to exhibit any modification in its cell proliferation level after both oestradiol and progesterone stimulation. Two cases definitively associated with an "ER-poor"/"PgR-poor" immunohistochemical phenotype nevertheless responded noticeably to the dynamic stimulation of their cell proliferation by oestradiol and progesterone. While our series of cases covers 14 patients only, it suffices to demonstrate the limits of ER and PgR determination in characterizing steroid hormone sensitivity in breast cancer. DISCUSSION: The present work therefore presents an in vitro approach to test growth regulation of human breast cancer by steroid hormones. The clinical value of the present approach should be further determined by showing that steroid hormone-induced modifications in cell proliferation level are actually associated with clinical response.

Grading dysplasia in colorectal adenomas by means of the quantitative binding pattern determination of Arachis hypogaea, Dolichos biflorus, Amaranthus caudatus, Maackia amurensis, and Sambucus nigra agglutinins.

The current study deals with the setting up of a new tool that enables the benign versus the malignant nature of colorectal adenomas to be determined accurately. The 2 objectives are to determine (1) whether adenomas should, or should not, be included in a 2- or a 3-tier grading system, and (2) whether severe dysplasias and carcinomas in situ share common or different biological characteristics. The levels of expression of different types of glycoconjugates were characterized in a series of 166 colorectal specimens, including 14 normal, 90 dysplastic, and 62 cancerous cases. The glycoconjugate expressions were demonstrated for 5 lectins, namely, Arachis hypogaea (PNA), Dolichos biflorus (DBA), Amaranthus caudatus (ACA), Maackia amurensis (MAA) and Sambucus nigra (SNA). The glycoconjugates demonstrated by these 5 lectins belong to the family of the Thomsen-Friedenreich antigens. The binding patterns of the 5 lectins were quantitatively determined by means of computer-assisted microscopy. The quantitative data were submitted to discriminant analyses. Our results show that the specific glycochemical staining patterns could be identified unambiguously and without misclassification between benign (normal and low dysplasia) and malignant (ie, either as moderate/severe dysplasia, carcinoma in situ, or cancer) cases. The data also strongly suggested that (1) dysplasias seem to be distinguishable in 2 instead of 3 groups, that is, low versus moderate/severe (high); and (2) moderate/severe dysplasias are biologically distinct from carcinomas in situ. The methodology developed can be applied directly in routine diagnosis to identify moderate/severe dysplasia specimens already exhibiting features common to carcinomas, and which therefore should be treated consistently in view of the fact that our data strongly suggest that most moderate/severe dysplasias are still benign, whereas carcinomas in situ are real carcinomatous lesions.

In vitro influence of lectins and neoglycoconjugates on the growth of three human sarcoma cell lines.

PURPOSE: The aim of our study is to investigate the in vitro effects of plant lectins, galectins and neoglycoconjugates on the proliferation of three human sarcoma cell lines. METHODS: Proliferation was assessed by means of the tetrazolium derivative reduction (MTT) assay. In addition, glycohistochemistry was used to make visible the plant-lectin-specific binding sites; the intensity of the lectin binding pattern was quantified by means of image analysis. RESULTS: Depending on the cell lines, the staining intensity and the percentage of labelled cells were different. With respect to growth modulation, the cell lines also responded differently to the probes used. Besides a predominant inhibitory effect elicited by the probes at 50 microg/ml, dose-dependent effects, including growth stimulation, were detectable in several instances. These effects relate to the animal galectins tested and several neoglycoconjugates, e.g. the lactose- and blood-group-A-trisaccharide-bearing probes. CONCLUSIONS: Endogenous lectins and lectin-reactive cellular glycoconjugates can apparently affect the regulation of the growth of human sarcoma cells. We suggest that these results are relevant for further histopathological monitoring in correlation with prognosis and in vitro assays to reveal possible clinical applications.

Characterization of biological features and chemosensitivity of a new experimental lung metastasis model originating from the MXT mouse mammary adenocarcinoma.

The present study shows how an original mouse metastatic lung model was established from the MXT mammary adenocarcinoma. This metastatic model was obtained by injecting the C/MET clone into the tail veins of B6D2F1 mice. The C/MET clone corresponds to one of eleven cell clones that were isolated in vitro from the MXT model. Of these 11 clones, only the C/MET leads to lung metastatic tumor development when injected i.v. into mice. Furthermore, the C/MET clone colonizes the lung only. The present data show that the C/MET metastatic model and the MXT parental line are weakly (if reference is made to the P388 leukemia model) sensitive to adriamycin, clyclophosphamide and etoposide. However, under specific experimental conditions, the chemosensitivity of the C/MET model can be significantly increased. The C/MET model therefore appears to be an interesting pharmacological tool to test new investigational agents with anti-tumor potentialities to lung metastases.

The influence of L-triiodothyronine, L-thyroxine, estradiol-17beta, the luteinizing-hormone-releasing hormone, the epidermal growth factor and gastrin on cell proliferation in organ cultures of 35 benign and 13 malignant human thyroid tumors.

PURPOSE: To characterize the influence of six factors on human thyroid tissues at the cell-proliferation level. These six factors were the epidermal growth factor (EGF), the luteinizing-hormone-releasing hormone (LHRH), triiodothyronine, thyroxine, estradiol and gastrin. METHODS: Forty-eight human thyroid specimens were obtained from surgical resection and maintained alive for 48 h ex vivo (in vitro) under organotypic culture conditions. These specimens comprised 35 benign cases (17 multinodular goiters and 18 adenomas) and 13 cancers. Cell proliferation in the control and treated conditions (at a 5 nM dose) was assessed by means of the thymidine labeling index, which enables the percentage of cells in the S phase of the cell cycle to be determined in accordance with autoradiographic procedures. RESULTS: The results show that, of the six factors tested here, EGF significantly (P < 0.05 to P < 0.001) increased cell proliferation in the greatest number of cancers as compared to what happened with the remaining five. Each of these six factors significantly increased or decreased proliferative cell activity in some 10%-30% of the cases under study. CONCLUSIONS: Triiodothyronine, thyroxine, LHRH and gastrin may increase or decrease cell proliferation in human thyroid tissues, whether benign or malignant, to the same extent as other hormones and/or growth factors such as thyrotropin, EGF, insulin-like growth factor 1, transforming growth factor beta1 and estradiol the effects of which on thyroid cell proliferation are already well documented in the literature.

In vitro motility evaluation of aggregated cancer cells by means of automatic image processing.

BACKGROUND: Set up of an automatic image processing based method that enables the motility of in vitro aggregated cells to be evaluated for a number of hours. METHODS: Our biological model included the PC-3 human prostate cancer cell line growing as a monolayer on the bottom of Falcon plastic dishes containing conventional culture media. Our equipment consisted of an incubator, an inverted phase contrast microscope, a Charge Coupled Device (CCD) video camera, and a computer equipped with an image processing software developed in our laboratory. This computer-assisted microscope analysis of aggregated cells enables global cluster motility to be evaluated. This analysis also enables the trajectory of each cell to be isolated and parametrized within a given cluster or, indeed, the trajectories of individual cells outside a cluster. RESULTS: The results show that motility inside a PC-3 cluster is not restricted to slight motion due to cluster expansion, but rather consists of a marked cell movement within the cluster. CONCLUSIONS: The proposed equipment enables in vitro aggregated cell motility to be studied. This method can, therefore, be used in pharmacological studies in order to select anti-motility related compounds. The compounds selected by the equipment described could then be tested in vivo as potential anti-metastatic.

Galectin fingerprinting in tumor diagnosis. Differential expression of galectin-3 and galectin-3 binding sites, but not galectin-1, in benign vs malignant uterine smooth muscle tumors.

Cell-matrix interactions are governed by a distinct set of proteins, with 2 nonintegrin laminin-binding proteins, galectin-1 and galectin-3, providing 1 aspect. The expression patterns of laminin and the 2 galectins and galectin binding sites were quantitatively determined by means of computer-assisted microscopy with the aim of differentiating between 16 leiomyomas and 10 leiomyosarcomas of the uterus. Three quantitative variables were computed for each of the 5 histochemical markers: labeling index, which describes the percentage of tissue area specifically stained by a given marker; mean optical density which reflects the concentration of the marker; and concentrational heterogeneity, which characterizes the degree of heterogeneity of the marker distribution in the tumor tissue areas. The results reveal evident differences in the galectin-3-related parameters in the 2 tumors groups. Whereas the concentration of galectin-3 binding sites was significantly (P = .01) weaker in the leiomyosarcomas than in the leiomyomas, the percentages of tumor tissue expressing galectin-3 (P = .02) and its binding sites (P = .002) were significantly higher in the leiomyosarcomas than in the leiomyomas. Although significantly (P = .02) higher, the concentration of laminin was more heterogeneously distributed (P = .01) in the leiomyosarcomas than in the leiomyomas. In contrast, the levels of expression of galectin-1 and its accessible binding sites remained similar for both the leiomyomas and the leiomyosarcomas. Finally we document how the levels of expression of galectin-3 and its binding sites can be of assistance in reliably differentiating leiomyomas from leiomyosarcomas.

Setting up of an original computer-assisted methodology to characterize in vitro drug-induced anti-angiogenic effects.

The development of angiogenesis within a tumor brings on a sequence of extremely complex molecular events. We have developed a methodology which enables a wide set of biological parameters to be quantitatively determined in the field of anti-angiogenesis pharmacology. This methodology which includes a video cell tracking device, is unique because it offers the possibility of evaluating the specific influence of a given compound with potential anti-angiogenic properties on cell cycle kinetics, cell death, global cell line growth, and cell motility. We chose TNP-470, a synthetic analogue of fumagilin, to test our methodology on HUVEC cell lines taken from various human umbilical cord veins. The experiments carried out with TNP-470 did not confirm all the data reported in the literature. Our results show that i) TNP-470 could be considered as a cytotoxic agent; ii) this compound had an apparently marginal cytostatic effect; and iii) it did not increase the apoptosis level. Our methodology also revealed that the HUVEC cell lines are very heterogeneous in terms of different biological parameters. This highlights the problem of the reproductibility of the result.

Gastrin inhibits motility, decreases cell death levels and increases proliferation in human glioblastoma cell lines.

Whether they are of low or high histopathological grade, human astrocytic tumors are characterized by a marked propensity to diffuse into large areas of normal brain parenchyma. This invasion relates mainly to cell motility, which enables individual cell migration to take place. The present study characterizes in vitro the gastrin-mediated effects on both the growth (cell proliferation vs. cell death) and motility dynamics of the human U87 and U373 glioblastoma cell lines. A computer-assisted phase-contrast microscope was used to track the number of mitoses versus cell deaths every 4 min over a 72-h period and so to quantitatively describe the trajectories of living U373 and U87 cells growing on plastic supports in culture media both with and without the addition of 0.1, 5, or 100 nM gastrin. While 5 or 100 nM gastrin only weakly (p < .05 to p < .01) increased cell proliferation in the U87 cell line and not in U373 one, it very significantly (p < .001) inhibited the amount of cell death at 5 and 100 nM in both the U87 and U373 lines. In addition, 5 nM gastrin markedly inhibited cell mobility in U87 (p < .00001) and U373 (p < .0001) glioblastoma models. All these data strongly suggest that gastrin plays a major role in the biological behavior of the in vitro U87 and U373 human glioblastoma cell lines in matters concerning their levels of cell motility and growth dynamics.

The in vitro influence of eight hormones and growth factors on the proliferation of eight sarcoma cell lines.

Little is known about the regulation of sarcoma proliferation by hormones and/or growth factors. We therefore characterised the in vitro proliferative influence on eight sarcoma cell lines of the platelet-derived growth factor, the insulin-like growth factor 1, triiodothyronine, the epidermal growth factor, the luteinising-hormone-releasing hormone, progesterone, gastrin and 17 beta-oestradiol. The influence of the different factors on the proliferation of sarcoma cell lines was measured by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test. Two culture media were studied: (1) a nutritionally poor medium containing 2% of fetal calf serum and (2) a nutritionally rich one containing 5% or 10% FCS both with and without the addition of non-essential amino acids. The results were analysed either by conventional statistical analyses or by a classification method based on a decision-tree approach developed in Machine Learning. This latter method was also compared to other classifiers (such as logistic regression and k nearest neighbours) with respect to its accuracy of classification. Monovariate statistical analysis showed that each of the eight cell lines exhibited sensitivity to at least one factor, and each factor significantly modified the proliferation of five or six of the eight cell lines under study. Of these eight lines one of fibrosarcoma origin was the most "factor-sensitive". Decision-tree-related data analysis enabled the specific pattern of factor sensitivity to be characterised for the three histological types of cell line under study. The effects of hormone and growth factors are significantly influenced by the type of culture medium used. The method used appeared to be an accurate classifier for the kind of data analysed. Sarcoma proliferation can be modulated, at least in vitro, by various hormones and growth factors, and the proliferation of each histopathological type exhibited a distinct sensitivity to different hormone and/or growth-factors.

Direct relationship between hormone sensitivity level and growth pattern. Evidence in 18 gastrointestinal neoplastic cell lines.

OBJECTIVE: We investigated whether a relationship exists in terms of growth pattern and hormone sensitivity in 18 gastrointestinal neoplastic cell lines. Hormones studied included gastrin, epidermal growth factor, estradiol and luteinizing hormone-releasing hormone. STUDY DESIGN: The growth patterns were assessed by means of computer-assisted microscope analysis of Feulgen-stained nuclei combined with the mathematical Delaunay triangulation and Voronoi paving techniques. This methodology enabled four variables characterizing the cell colony patterns to be computed. The information contributed by these variables was analyzed by means of discriminant analysis and the decision tree technique. RESULTS: Each phenotype (sensitivity level) exhibited distinct growth pattern (or cell colony) characteristics in the case of each hormone and/or growth factor under study. Furthermore, the sensitivity of the gastrointestinal cell lines to a given hormone (or growth factor) appeared to be peculiar to the hormone (or growth factor). CONCLUSION: A direct relationship seems to exist between growth pattern and hormone sensitivity levels in gastrointestinal cancers, particularly colorectal.

Growth inhibition of human in vitro and mouse in vitro and in vivo mammary tumor models by retinoids in comparison with tamoxifen and the RU-486 anti-progestagen.

Retinoids constitute a very promising class of agents for the chemoprevention or treatment of breast cancer. These retinoids exert their biological activity through two distinct classes of retinoic acid (RA) receptors (R), the RAR isotypes (alpha, beta, and gamma) and the three RXR isotypes (alpha, beta, and gamma) and their numerous isoforms which bind as RXR/RAR heterodimers to the polymorphic cis-acting response elements of RA target genes. With respect to these numerous receptor sub-types, the retinoid-induced effects at the biological level include marked modifications with respect to both cell proliferation and cell death (apoptosis), and also in the induction of differentiation processes. The present study aims to characterize the effect which four retinoids (TTNPB, 9-cis-RA, LGD 1069, 4-HPR) with distinct RAR/RXR binding properties induced on various in vitro and in vivo mouse and human breast cancer models. The experiments with the retinoids were carried out in comparison with the anti-estrogen tamoxifen and the anti-progestagen RU-486 compounds. The results show that the 6 compounds under study were markedly more efficient in terms of growth inhibition in the human T-47D cell line when maintained under anchorage-independent culture conditions than when maintained under anchorage-dependent ones. While RU-486 exhibited a weak statistically significant (p < 0.05) influence on the growth of the T-47D stem cells, tamoxifen had a marked inhibitory influence on the growth of these cells. Of the four retinoids, 4-HPR was the least effective since the lowest doses tested (1 and 0.1 nM) exhibited no statistically (p > 0.05) significant influence on the growth of the stem cells. The most efficient retinoid was TTNPB. It was only at the highest dose (10 microM) that tamoxifen and RU-486 showed a weak inhibitory influence on the growth of the T-47D non-stem cells while all 4 retinoids exerted a significant inhibitory influence on the growth of these non-stem cells, with 4-HPR being the most efficient (P < 0.001) at the highest dose, but ineffective (P > 0.05) at the lowest. Tamoxifen and TTNPB were tested in vivo on hormone-sensitive (HS) and hormone-insensitive (HI) strains of the MXT murine mammary carcinoma. While TTNPB appeared to be equally efficient in terms of growth inhibition in both MXT-HS and MXT-HI models, tamoxifen had only a marginal inhibitory influence on the growth of the MXT-HI strain but did inhibit growth in the case of the MXT-HS one. TTNPB was markedly more efficient than tamoxifen in terms of both inhibiting the cell proliferation level (measured by means of computer-assisted microscopy applied to Feulgen-stained nuclei, a method which enables the percentage of cells in the S phase of the cell cycle to be determined) and triggering cell death (measured by means of the determination of the transglutaminase activity) in both the MXT-HI and MXT-HS models. The very significant TTNPB-induced inhibition of the macroscopic MXT-HS growth rate relates to the triggering of cell death (apoptosis) rather than to an inhibition of cell proliferation. All these results clearly indicate that retinoids are very efficient agents against breast cancer, at least as efficient as tamoxifen.