The molecular landscape and associated clinical experience in infant medulloblastoma: prognostic significance of second-generation subtypes.

AIMS: Biomarker-driven therapies have not been developed for infant medulloblastoma (iMB). We sought to robustly sub-classify iMB, and proffer strategies for personalized, risk-adapted therapies. METHODS: We characterized the iMB molecular landscape, including second-generation subtyping, and the associated retrospective clinical experience, using large independent discovery/validation cohorts (n = 387). RESULTS: iMBGrp3 (42%) and iMBSHH (40%) subgroups predominated. iMBGrp3 harboured second-generation subtypes II/III/IV. Subtype II strongly associated with large-cell/anaplastic pathology (LCA; 23%) and MYC amplification (19%), defining a very-high-risk group (0% 10yr overall survival (OS)), which progressed rapidly on all therapies; novel approaches are urgently required. Subtype VII (predominant within iMBGrp4 ) and subtype IV tumours were standard risk (80% OS) using upfront CSI-based therapies; randomized-controlled trials of upfront radiation-sparing and/or second-line radiotherapy should be considered. Seventy-five per cent of iMBSHH showed DN/MBEN histopathology in discovery and validation cohorts (P < 0.0001); central pathology review determined diagnosis of histological variants to WHO standards. In multivariable models, non-DN/MBEN pathology was associated significantly with worse outcomes within iMBSHH . iMBSHH harboured two distinct subtypes (iMBSHH-I/II ). Within the discriminated favourable-risk iMBSHH DN/MBEN patient group, iMBSHH-II had significantly better progression-free survival than iMBSHH-I , offering opportunities for risk-adapted stratification of upfront therapies. Both iMBSHH-I and iMBSHH-II showed notable rescue rates (56% combined post-relapse survival), further supporting delay of irradiation. Survival models and risk factors described were reproducible in independent cohorts, strongly supporting their further investigation and development. CONCLUSIONS: Investigations of large, retrospective cohorts have enabled the comprehensive and robust characterization of molecular heterogeneity within iMB. Novel subtypes are clinically significant and subgroup-dependent survival models highlight opportunities for biomarker-directed therapies.

Participation of vitamin D-upregulated protein 1 (TXNIP)-ASK1-JNK1 signalosome in the enhancement of AML cell death by a post-cytotoxic differentiation regimen.

Standard therapy for Acute Myeloid Leukemia (AML) is rarely curative, and several suggested improvements have had little success so far. We have reported that in an in vitro model of a potential therapeutic regimen for AML, the activity of cytarabine (AraC) is enhanced by a sequential treatment with a combination of the vitamin D2 analog Doxercalciferol (D2) and the plant-derived antioxidant carnosic acid (CA). Importantly, the enhancement occurred selectively in patient-derived AML blasts, but not in the normal bone marrow cells. We now demonstrate that TXNIP, previously known as Vitamin D up-regulated protein 1 (VDUP1) [PMID 808674] plays a part in signaling cell death (CD) in this regimen. This is shown by the reduced CD when TXNIP protein levels are decreased by the CRISPR/CAS9 or RNAi technology. Further, we show that direct activation of ASK1 kinase by TXNIP is required for the optimal transmission of the CD signal to apoptotic machinery, regulated by JNK and BIM. These studies provide a rationale for a projected clinical trial of this vitamin D-based new therapeutic regimen for AML.

Antitumor activity of ethanol extract from Hippophae rhamnoides L. leaves towards human acute myeloid leukemia cells in vitro.

We studied the effects of ethanol extract from Hippophae rhamnoides L. leaves on the growth and differentiation of human acute myeloid leukemia cells (KG-1a, HL60, and U937). The extract of Hippophae rhamnoides L. leaves inhibited cell growth depending on the cell strain and extract dose. In a high concentration (100 µg/ml), the extract also exhibited a cytotoxic effect on HL60 cells. Hippophae rhamnoides L. leaves extract did not affect cell differentiation and did not modify the differentiating effect of calcitriol, active vitamin D metabolite. Inhibition of cell proliferation was paralleled by paradoxical accumulation of phase S cells (synthetic phase) with a reciprocal decrease in the count of G1 cells (presynthetic phase). The extract in a concentration of 100 µg/ml induced the appearance of cells with a subdiploid DNA content (sub-G1 phase cells), which indicated induction of apoptosis. The antiproliferative effect of Hippophae rhamnoides L. extract on acute myeloid leukemia cells was at least partially determined by activation of the S phase checkpoint, which probably led to deceleration of the cell cycle and apoptosis induction.

Distribution of antigen-presenting cells CD68 in papillomavirus infection in the skin.

We analyzed local reactions of immune homeostasis in the human skin, in particular, effector immune cells CD68 responsible for antigen presentation, during human papillomavirus infection. Under conditions of long-term papillomavirus infection, CD68 markers were identifi ed only in the connective tissue of the skin (derma) and were completely absent in the epidermis, where they were found during physiological and reparative regeneration after thermal injury. We concluded that hypertrophy of the epidermis and connective tissue of the dermal papillary layer in human papillomavirus infection is related to the absence of CD68 immune cells in the epithelial plate and their accumulation in the connective tissue adjacent to the basement membrane of the epidermis. The possibility of epithelium contamination with the virus depends on local immune homeostasis. Therefore, induction of proper CD68 distribution in appropriate structures can contribute to normalization of epithelial-connective tissue interactions.

Differential enhancement of leukaemia cell differentiation without elevation of intracellular calcium by plant-derived sesquiterpene lactone compounds.

BACKGROUND AND PURPOSE: All-trans retinoic acid (ATRA) induces complete remission in a majority of acute promyelocytic leukaemia patients, but resistance of leukaemic cells to ATRA and its toxicity, such as hypercalcaemia, lead to a limitation of treatment. Therefore, combination therapies with differentiation-enhancing agents at non-toxic concentrations of ATRA may overcome its side effects. Here, we investigated the effect of plant-derived sesquiterpene lactone compounds and their underlying mechanisms in ATRA-induced differentiation of human leukaemia HL-60 cells. EXPERIMENTAL APPROACH: HL-60 cells were treated with four sesquiterpene lactones (helenalin, costunolide, parthenolide and sclareolide) and cell differentiation was determined by NBT reduction, Giemsa and cytofluorometric analyses. Signalling pathways were assessed by western blotting, gel-shift assay and kinase activity determinations and intracellular calcium levels were determined using a calcium-specific fluorescent probe. KEY RESULTS: Helenalin, costunolide and parthenolide, but not sclareolide, increased ATRA-induced HL-60 cell differentiation into a granulocytic lineage. Signalling kinases PKC and ERK were involved in the ATRA-induced differentiation enhanced by all of the effective sesquiterpene lactones, but JNK and PI3-K were involved in the ATRA-induced differentiation enhanced by costunolide and parthenolide. Enhancement of cell differentiation closely correlated with inhibition of NF-kappaB DNA-binding activity by all three effective compounds. Importantly, enhancement of differentiation induced by 50 nM ATRA by the sesquiterpene lactones was not accompanied by elevation of basal intracellular calcium concentrations. CONCLUSIONS AND IMPLICATIONS: These results indicate that plant-derived sesquiterpene lactones may enhance ATRA-mediated cell differentiation through distinct pathways.

Carnosic acid and promotion of monocytic differentiation of HL60-G cells initiated by other agents.

BACKGROUND: Carnosic acid is a plant-derived polyphenol food preservative with chemoprotective effects against carcinogens when tested in animals. Recently, we showed that carnosic acid potentiates the effects of 1alpha,25-dihydroxyvitamin D3 (1alpha,25[OH]2D3) and of all-trans-retinoic acid (ATRA) on differentiation of human leukemia cells. We now examine the mechanisms associated with carnosic acid-induced enhancement of cell differentiation (in subline HL60-G) initiated by 1alpha,25(OH)2D3, ATRA, or 12-O-tetradecanoylphorbol-13-acetate (TPA). METHODS: We evaluated monocytic differentiation markers (CD11b, CD14, and monocytic serine esterase), cell cycle parameters, and cell proliferation rates after treatment of cells with different agents with or without carnosic acid. We also assessed the abundance of the vitamin D receptor (VDR), retinoid X receptor (RXR)-alpha, retinoic acid receptor (RAR)-alpha, and cell cycle-associated proteins by immunoblot analysis (p27, early growth response gene [EGR]-1, and p35Nck5a), the expression of corresponding genes by reverse transcription-polymerase chain reaction (RT-PCR), and the activity of VDR by electrophoretic mobility shift analysis. The two-sided nonparametric Kruskal-Wallis one-way analysis-of-variance test with Dunn's adjustment was used for statistical analyses. RESULTS: Monocytic differentiation induced by low (1 nM) concentrations of 1alpha,25(OH)2D3, ATRA, or TPA was enhanced by carnosic acid (10 microM), as shown by the increased expression of monocytic serine esterase (P<.001, P<.001, and P =.043, respectively) and of CD11b (P =.008, P =.046, and P =.041, respectively). Increased expression of CD14 was seen only for 1alpha,25(OH)2D3 and ATRA (P =.009 and P =.048, respectively) and also for several cell cycle-associated proteins. Carnosic acid in combination with 1alpha,25(OH)2D3 and ATRA resulted in decreased cell proliferation and blocked the cell cycle transition from G1 to S phase (P<.05). Carnosic acid alone increased the expression of VDR and RXR-alpha, but the expression was greatly enhanced in the presence of 1alpha,25(OH)2D3 and ATRA. In combination with TPA, carnosic acid potentiated the expression of VDR and RAR-alpha. CONCLUSION: Carnosic acid enhances a program of gene expression consistent with 1alpha,25(OH)2D3-, ATRA-, or TPA-induced monocytic differentiation of HL60-G cells.

Effects of acyclo-retinoic acid and lycopene on activation of the retinoic acid receptor and proliferation of mammary cancer cells.

The biochemical mechanisms underlying the inhibitory effects of lycopene, the main tomato carotenoid, on the growth of cancer cells are largely unknown. It has been hypothesized that lycopene derivatives may act as ligands for a nuclear receptor in analogy to retinoic acid, the hormone derived from beta-carotene. The inhibition of human mammary cancer (MCF-7) cell growth and the transactivation of the retinoic acid receptor (RAR) reporter gene by synthetic acyclo-retinoic acid, the open chain analog of retinoic acid, was compared to the effects of lycopene and retinoic acid in the same systems. Acyclo-retinoic acid activated the DR-5 retinoic acid response element with a approximately 100-fold lower potency than retinoic acid. This effect was independent of cotransfection with the RARalpha receptor. Lycopene exhibited only very modest activity in this system. In contrast to the results from the transactivation studies, acyclo-retinoic acid, retinoic acid, and lycopene inhibited cell growth with a similar potency. Preincubation with each of the three compounds slowed down cell cycle progression from G1 to S phase. In summary, acyclo-retinoic acid inhibited cancer cell growth and interacted with RAR. However, it exhibited low affinity for RAR and a correspondingly low efficacy in activating this receptor, indicating that RAR does not mediate the growth inhibitory effect of the compound. In addition, the concentrations of acyclo-retinoic acid and of lycopene required for inducing inhibition of cell growth were similar, suggesting that acyclo-retinoic acid is unlikely to be the active metabolite of lycopene.

Lycopene inhibition of cell cycle progression in breast and endometrial cancer cells is associated with reduction in cyclin D levels and retention of p27(Kip1) in the cyclin E-cdk2 complexes.

Numerous studies have demonstrated the anticancer activity of the tomato carotenoid, lycopene. However, the molecular mechanism of this action remains unknown. Lycopene inhibition of human breast and endometrial cancer cell growth is associated with inhibition of cell cycle progression at the G(1) phase. In this study we determined the lycopene-mediated changes in the cell cycle machinery. Cells synchronized in the G(1) phase by serum deprivation were treated with lycopene or vehicle and restimulated with 5% serum. Lycopene treatment decreased serum-induced phosphorylation of the retinoblastoma protein and related pocket proteins. This effect was associated with reduced cyclin-dependent kinase (cdk4 and cdk2) activities with no alterations in CDK protein levels. Lycopene caused a decrease in cyclin D1 and D3 levels whereas cyclin E levels did not change. The CDK inhibitor p21(Cip1/Waf1) abundance was reduced while p27(Kip1) levels were unaltered in comparison to control cells. Serum stimulation of control cells resulted in reduction in the p27 content in the cyclin E--cdk2 complex and its accumulation in the cyclin D1--cdk4 complex. This change in distribution was largely prevented by lycopene treatment. These results suggest that lycopene inhibits cell cycle progression via reduction of the cyclin D level and retention of p27 in cyclin E--cdk2, thus leading to inhibition of G(1) CDK activities.

Carnosic acid inhibits proliferation and augments differentiation of human leukemic cells induced by 1,25-dihydroxyvitamin D3 and retinoic acid.

Carnosic acid, the polyphenolic diterpene derived from rosemary, is a strong dietary antioxidant that exhibits antimutagenic properties in bacteria and anticarcinogenic activity in various cell and animal models. In the present study, we show that carnosic acid (2.5-10 microM) inhibits proliferation of HL-60 and U937 human myeloid leukemia cells (half-maximal inhibitory concentration = 6-7 microM) without induction of apoptotic or necrotic cell death. Growth arrest occurred concomitantly with a transient cell cycle block in the G1 phase, which was accompanied by an increase in the immunodetectable levels of the universal cyclin-dependent kinase inhibitors p21WAFI and p27Kipl. Carnosic acid caused only a marginal induction of differentiation, as monitored by the capacity to generate superoxide radicals and the expression of cell surface antigens (CD11b and CD14) and receptors for the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine. However, at low concentrations, this polyphenol substantially augmented (100- to 1,000-fold) the differentiating effects of 1,25-dihydroxyvitamin D3 and all-trans retinoic acid. Furthermore, such combinations of carnosic acid and any of these differentiation inducers synergistically inhibited proliferation and cell cycle progression. These results indicate that carnosic acid is capable of antiproliferative action in leukemic cells and can cooperate with other natural anticancer compounds in growth-inhibitory and differentiating effects.

Lycopene interferes with cell cycle progression and insulin-like growth factor I signaling in mammary cancer cells.

Recent studies have shown that high insulin-like growth factor I (IGF-I) blood level is a risk factor in breast and prostate cancer. The aim of this study was to determine whether the mitogenic activity of IGF-I in mammary cancer cells can be reduced by the dietary carotenoid lycopene. The anticancer activity of lycopene, the major tomato carotenoid, has been suggested by in vitro, in vivo, and epidemiological studies. Growth stimulation of MCF7 mammary cancer cells by IGF-I was markedly reduced by physiological concentrations of lycopene. The inhibitory effects of lycopene on MCF7 cell growth were not accompanied by apoptotic or necrotic cell death, as determined by annexin V binding to plasma membrane and propidium iodide staining of nuclei in unfixed cells. Lycopene treatment markedly reduced the IGF-I stimulation of tyrosine phosphorylation of insulin receptor substrate 1 and binding capacity of the AP-1 transcription complex. These effects were not associated with changes in the number or affinity of IGF-I receptors, but with an increase in membrane-associated IGF-binding proteins, which were previously shown in different cancer cells to negatively regulate IGF-I receptor activation. The inhibitory effect of lycopene on IGF signaling was associated with suppression of IGF-stimulated cell cycle progression of serum-starved, synchronized cells. Moreover, in cells synchronized by mimosine treatment, lycopene delayed cell cycle progression after release from the mimosine block. Collectively, the above data suggest that the inhibitory effects of lycopene on MCF7 cell growth are not due to the toxicity of the carotenoid but, rather, to interference in IGF-I receptor signaling and cell cycle progression.

Effect of purified allicin, the major ingredient of freshly crushed garlic, on cancer cell proliferation.

The diverse health benefit effects of garlic include its anticancer activity. However, very little is known about such activity of isolated garlic compounds, among which allicin (the major ingredient of crushed garlic) has been the least studied. The aim of this work was to determine whether pure allicin exhibits the antiproliferative effect reported for garlic in in vitro models. Allicin, but not its precursor alliin, inhibited proliferation of human mammary (MCF-7), endometrial (Ishikawa), and colon (HT-29) cancer cells (50% inhibitory concentration = 10-25 microM). Two of three tested primary lines of human fibroblasts displayed a similar response to allicin (50% inhibitory concentration = 16-40 microM), whereas the third line was almost unaffected by this compound. The pure allicin and water extract of garlic powder with equivalent allicin concentrations displayed a similar potency, suggesting that allicin is responsible for the antiproliferative effect of the extract. The growth inhibition was accompanied by accumulation of cells in the G0/G1 and G2/M phases of the cell cycle (MCF-7 cells) and not by a significant increase in cell death. Allicin caused a transient drop in the intracellular glutathione (GSH) level, the magnitude and kinetics of which significantly varied depending on cell type. The extent of the decrease in GSH levels correlated well (r = 0.75) with the growth inhibitory activity of allicin. On the basis of these findings, we suggest that allicin plays a major role in the antiproliferative effect of water-soluble garlic preparations and that this effect may be attributed to the ability of allicin to transiently deplete the intracellular GSH level.

Lycopene and 1,25-dihydroxyvitamin D3 cooperate in the inhibition of cell cycle progression and induction of differentiation in HL-60 leukemic cells.

Lycopene, the major tomato carotenoid, has been found to inhibit proliferation of several types of cancer cells, including those of breast, lung, and endometrium. By extending the work to the HL-60 promyelocytic leukemia cell line, we aimed to evaluate some mechanistic aspects of this effect. Particularly, the possibility was examined that the antiproliferative action of the carotenoid is associated with induction of cell differentiation. Lycopene treatment resulted in a concentration-dependent reduction in HL-60 cell growth as measured by [3H]thymidine incorporation and cell counting. This effect was accompanied by inhibition of cell cycle progression in the G0/G1 phase as measured by flow cytometry. Lycopene alone induced cell differentiation as measured by phorbol ester-dependent reduction of nitro blue tetrazolium and expression of the cell surface antigen CD14. Results of several recent intervention studies with beta-carotene, which have revealed no beneficial effects of this carotenoid, suggest that a single dietary component cannot explain the anticancer effect of diets rich in vegetables and fruits. Thus another goal of our study was to examine whether lycopene has the ability to synergize with other natural anticancer compounds, such as 1,25-dihydroxyvitamin D3, which when used alone are therapeutically active only at high and toxic concentrations. The combination of low concentrations of lycopene with 1,25-dihydroxyvitamin D3 exhibited a synergistic effect on cell proliferation and differentiation and an additive effect on cell cycle progression. Such synergistic antiproliferative and differentiating effects of lycopene and other compounds found in the diet and in plasma may suggest the inclusion of the carotenoid in the diet as a cancer-preventive measure.

Membrane-associated insulin-like growth factor-binding protein-3 inhibits insulin-like growth factor-I-induced insulin-like growth factor-I receptor signaling in ishikawa endometrial cancer cells.

The function of cell surface-associated insulin-like growth factor-binding proteins (IGFBPs) is controversial. Both inhibition and facilitation of IGF action as well as IGF-independent effects have been reported. We examined the influence of endogenous cell surface-associated IGFBPs on IGF-I receptor (IGF-IR) function in Ishikawa endometrial cancer cells by comparing the effects of IGF-I and its truncated analog des-(1-3)-IGF-I on several components of the IGF-IR signal transduction pathway in the absence of significant amounts of soluble IGFBPs. IGF-I and des-(1-3)-IGF-I are known to have similar affinities for IGF-IR, although the affinity of des-(1-3)-IGF-I for IGFBPs is greatly reduced. Here we show that the two ligands were equipotent not only in IGF-IR binding but also in receptor activation in NIH 3T3 cells overexpressing IGF-IR and possessing a relatively small number of cell surface-associated IGFBPs. In contrast, des-(1-3)-IGF-I manifested a remarkably higher potency as compared with IGF-I in inducing short and middle term cellular responses in IGF-IR-transfected Ishikawa endometrial cancer cells possessing a high number of both the receptor and the cell membrane-bound IGFBP-3. Thus, this difference in the effects of IGF-I and des-(1-3)-IGF-I can be attributed to the attenuation of IGF-I-mediated IGF-IR signaling by membrane-bound IGFBP-3.

Na(+)-K(+)-ATPase in frog esophagus mucociliary cell membranes: inhibition by protein kinase C activation.

We examined protein kinase C (PKC)-dependent regulation of Na(+)-K(+)-ATPase in frog mucociliary cells. Activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) or 1,2-dioctanoyl-sn-glycerol (diC8) either in intact cells or isolated membranes resulted in a specific inhibition of Na(+)-K(+)-ATPase activity by approximately 25-45%. The inhibitory effects in membranes exhibited time dependence and dose dependence [half-maximal inhibition concentration (IC50) = 0.5 +/- 0.1 nM and 2.4 +/- 0.2 microM, respectively, for TPA and diC8] and were not influenced by Ca2+. Analysis of the ouabain inhibition pattern revealed the presence of two Na(+)-K(+)-ATPase isoforms with IC50 values for cardiac glycoside of 2.6 +/- 0.8 nM and 409 +/- 65 nM, respectively. Most importantly, the isoform possessing a higher affinity for ouabain was almost completely inhibited by TPA, whereas its counterpart was hardly sensitive to the PKC activator. The results suggest that, in frog mucociliary cells, PKC regulates Na(+)-K(+)-ATPase and that this action is related to the specific Na(+)-K(+)-ATPase isoform.

Stimulation of endometrial cancer cell growth by tamoxifen is associated with increased insulin-like growth factor (IGF)-I induced tyrosine phosphorylation and reduction in IGF binding proteins.

A significant increase in endometrial cancer incidence in tamoxifen-treated breast cancer patients has been reported in many recent studies. The major growth stimulators of endometrial tumors are estrogens, but paradoxically, tamoxifen, a known antiestrogen, also stimulates their growth. The mode of action of estrogen can be partially explained by the modulation of insulin-like growth factor (IGF) autocrine or paracrine action. The purpose of the present study was to examine the involvement of the IGF system in the tamoxifen-stimulated growth of Ishikawa endometrial cancer cells by quantitating the IGF-I receptors and their phosphorylation, as well as membrane associated and secreted IGF-binding proteins (IGFBPs). Tamoxifen did not affect the number or affinity of IGF-I receptors. On the other hand, tamoxifen, similar to estradiol, increased IGF-I-stimulated tyrosine phosphorylation of cellular substrates. In contrast, in MCF-7 mammary cancer cells, tamoxifen reduced IGF-induced tyrosine phosphorylation in the presence of estradiol. The pure antiestrogen LY156758 did not affect Ishikawa basal cell growth but inhibited estradiol- and tamoxifen-induced growth. Growth inhibition by LY156758 of tamoxifen and estradiol-stimulated cells was accompanied by a corresponding inhibition of IGF-stimulated tyrosine phosphorylation. Tamoxifen caused a 3-fold decrease in membrane-associated IGFBPs. Moreover, a reduction in soluble IGFBPs was also observed, making the IGF peptides more available to the receptors. A parallel decrease in IGFBP-3 mRNA was also detected. These experiments suggest that tamoxifen, like estradiol, directly sensitizes endometrial cancer cells to the effects of IGFs that act through the type I receptor. Furthermore, the decrease in IGFBPs and the increase in tyrosine phosphorylation in the presence of tamoxifen provides a molecular mechanism that accounts for the uterotropic effects that are seen with tamoxifen therapy.

The assembly of neutrophil NADPH oxidase: effects of mastoparan and its synthetic analogues.

Detergent-mediated activation of the phagocyte superoxide-generating NADPH oxidase requires the participation of at least four proteins: the membrane-bound heterodimeric cytochrome b558 and three cytosolic components, p47-phox, p67-phox and a Rac1/Rac2 protein. Peptides corresponding to sequences of different subunits of NADPH oxidase have been used as probes of the mechanism and sequence of assembly of the active complex. In the present study effects of mastoparans on activation of NADPH oxidase were investigated. Mastoparans are wasp venom cationic amphiphilic tetradecapeptides capable of modulation of various cellular activities. Natural mastoparans, as well as several synthetic mastoparan analogues, unrelated to oxidase components, blocked activation of the oxidase in the cell-free system (EC50 = 1.5 microM) and in guanosine 5'-[gamma-thio]triphosphate (GTP[S])/ATP-stimulated neutrophils permeabilized with streptolysin O. In the cell-free system the effect was not relieved by raising the detergent concentration and could not be ascribed to changes in critical micellar concentration values of the activating SDS or arachidonate. Chromatography of neutrophil cytosol on an immobilized mastoparan column suggested interaction of cytosolic p47-phox and p67-phox with the peptide. In spite of this interaction mastoparan did not interfere with translocation of p47-phox and p67-phox to the cell membranes.

Lycopene is a more potent inhibitor of human cancer cell proliferation than either alpha-carotene or beta-carotene.

The antiproliferative properties of lycopene, the major tomato carotenoid, were compared with those of alpha- and beta-carotene. Lycopene, delivered in cell culture medium from stock solutions in tetrahydrofuran, strongly inhibited proliferation of endometrial (Ishikawa), mammary (MCF-7), and lung (NCI-H226) human cancer cells with half-maximal inhibitory concentration of 1-2 microM; alpha- and beta-carotene were far less effective inhibitors. For example, in Ishikawa cells, a 4-fold higher concentration of alpha-carotene or a 10-fold higher concentration of beta-carotene was needed for the same order of growth suppression. The inhibitory effect of lycopene was detected after 24 hours of incubation, and it was maintained for at least three days. In contrast to cancer cells, human fibroblasts were less sensitive to lycopene, and the cells gradually escaped growth inhibition over time. In addition to its inhibitory effect on basal endometrial cancer cell proliferation, lycopene also suppressed insulin-like growth factor-I-stimulated growth. Insulin-like growth factors are major autocrine/paracrine regulators of mammary and endometrial cancer cell growth. Therefore, lycopene interference in this major autocrine/paracrine system may open new avenues for research on the role of lycopene in the regulation of endometrial cancer and other tumors.

Components of the IGF system mediate the opposing effects of tamoxifen on endometrial and breast cancer cell growth.

The involvement of the IGF system in the growth regulation of hormone-dependent (e.g. endometrial and breast) cancer cells was studied. We chose two opposing effects of tamoxifen: the paradoxical stimulation of Ishikawa endometrial cancer cells growth and its inhibitory effect on MCF-7 mammary cancer cells. The results clearly confirm our working hypothesis that the IGF system is involved in growth regulation of these cancer cells irrespective of the direction of the drug effect. The following parameters of the IGFs system were studied: IGF-I receptors, IGF-I stimulated protein tyrosine phosphorylation, and membrane-associated and secreted IGF-binding proteins (IGFBPs). In Ishikawa cells, tamoxifen, similar to estradiol, increased IGF-I stimulated tyrosine phosphorylation of cellular substrates in accordance with its effect on cell growth. This effect of tamoxifen was inverted in MCF-7 cells. Tamoxifen did not affect the number or affinity of IGF-I receptors in both Ishikawa and MCF-7 cells, however, it caused a three-fold decrease in membrane-associated IGFBPs in the endometrial cells but an increase in these proteins in breast cancer cells. Similar but much less pronounced changes in soluble IGFBPs were observed. Our results indicate that the opposing growth effects of tamoxifen an endometrial and mammary cancer cells are associated with modulation of the IGF system components, mainly with reciprocal changes in membrane-associated IGFBPs.

Selective effects of mastoparan analogs: separation of G-protein-directed and membrane-perturbing activities.

Mastoparan (MP), a wasp venom peptide, is known both to interact with G-proteins and to alter membrane structure and function. To determine the structural requirements for these two aspects of MP action, we constructed several analogs of the peptide and characterized them using Swiss 3T3 cell membranes. The effects of these peptides were measured on: i) G-protein-mediated stimulation of phospholipase-C activity by GTP gamma S and bombesin and ii) the membrane enzyme activities, calcium-activated phospholipase-C and Na,K-ATPase. MP strongly inhibited all the above activities and caused membrane permeabilization. Substitution of one Lys residue by Gly at either the N- or C-terminal of the MP molecule resulted in peptides which selectively inhibited G-protein stimulated phospholipase-C with no or very slight membrane-perturbing effects. Introduction of additional Lys residues to MP led to the opposite effect. Thus, G-protein modulating and membrane disrupting actions of MP appear to be not necessarily linked, and may be separated.