FOXL2-induced follistatin attenuates activin A-stimulated cell proliferation in human granulosa cell tumors

Human granulosa cell tumors (GCTs) are rare, and their etiology remains largely unknown. Recently, the FOXL2 402C > G (C134W) mutation was found to be specifically expressed in human adult-type GCTs; however, its function in the development of human GCTs is not fully understood. Activins are members of the transforming growth factor-beta superfamily, which has been shown to stimulate normal granulosa cell proliferation; however, little is known regarding the function of activins in human GCTs. In this study, we examined the effect of activin A on cell proliferation in the human GCT-derived cell line KGN. We show that activin A treatment stimulates KGN cell proliferation. Treatment with the activin type I receptor inhibitor SB431542 blocks activin A-stimulated cell proliferation. In addition, our results show that cyclin D2 is induced by treatment with activin A and is involved in activin A-stimulated cell proliferation. Moreover, the activation of Smad signaling is required for activin A-induced cyclin D2 expression. Finally, we show that the overexpression of the wild-type FOXL2 but not the C134W mutant FOXL2 induced follistatin production. Treatment with exogenous follistatin blocks activin A-stimulated cell proliferation, and the overexpression of wild-type FOXL2 attenuates activin A-stimulated cell proliferation. These results suggest that FOXL2 may act as a tumor suppressor in human adult-type GCTs by inducing follistatin expression, which subsequently inhibits activin-stimulated cell proliferation.     

Inhibition of human tumor cell proliferation by novel anthraquinones from daylilies

Daylilies (Hemerocallis) are used medicinally in eastern Asia and extracts of the plant had been shown to inhibit cell proliferation and induce cancer cells to undergo differentiation. In our studies of the constituents of Hemerocallis fulva var. 'Kwanzo' roots, we isolated a series of new [kwanzoquinones A (1), B (2), C (4), D (5), E (6), F (7), G (9)] and known [2-hydroxychrysophanol (3) and rhein (8)] anthraquinones. These compounds were tested in order to determine their potential roles as cancer cell growth inhibitors. Kwanzoquinones A-C and E, kwanzoquinone A and B monoacetates (1a and 2a), 2-hydroxychrysophanol, and rhein inhibited the proliferation of human breast, CNS, colon, and lung cancer cells with GI50 values between 1.8 to 21.1 microg/mL. However, upon exposure of the cancer cells to the GI50 concentrations of the bioactive anthraquinones, most of the cancer cell lines exhibited higher than anticipated levels of cell viability. Co-incubation of the anthraquinones with vitamins C and E increased the viability of breast cancer cells. In contrast, vitamins C and E potentiated the cytotoxic effects of the anthraquinones against the colon cancer cells. None of the anthraquinones inhibited the activity of topoisomerase.     

Interferon-alpha inhibits proliferation in human T lymphocytes by abrogation of interleukin 2-induced changes in cell cycle-regulatory proteins.

IFN-alpha exerts prominent regulatory functions on the immune system. One such effect is the inhibition of proliferation of in vitro stimulated T lymphocytes. The exact physiological function of this activity is not known, but it has been implicated in the antiviral effects of IFN, its antitumor action in T-cell malignancies, and the regulation of the in vivo T-cell response. Here, we have investigated the mechanism underlying the IFN-alpha-mediated growth inhibition of normal human PHA- and IL-2-stimulated T lymphocytes by an analysis of how IFN-alpha treatment influences known molecular events that normally accompany the transition from quiescence to proliferation in these cells. IFN-alpha treatment was found to profoundly block S-phase entry of stimulated T lymphocytes. This correlated with a strong inhibition of IL-2-induced changes in G1-regulatory proteins, including the prevented up-regulation of G1 cyclins and cyclin-dependent kinases as well as an abrogation of mitogen-induced reduction of p27Kip1 levels. This latter effect was due to a maintained stability of the p27Kip1 protein in the IFN-alpha-treated cells. In line with these findings, phosphorylation of the pocket proteins was abrogated in IFN-alpha-treated cells. Furthermore, our data indicate that IFN-alpha has selective effects on the pathways that emerge from the IL-2 receptor because IFN-alpha treatment does not block IL-2-induced up-regulation of c-myc or Cdc25A.     

Suppression of IL-2-induced T cell proliferation and phosphorylation of STAT3 and STAT5 by tumor-derived TGF beta is reversed by IL-15.

IL-2 responses are susceptible to suppression by TGFbeta, a cytokine widely implicated in suppression of inflammatory responses and secreted by many different tumor cell types. There have been conflicting reports regarding inhibition of IL-2-induced STAT3 and STAT5 phosphorylation by TGFbeta and subsequent suppression of immune responses. Using TGFbeta-producing multiple myeloma tumor cells we demonstrate that tumor-derived TGFbeta can block IL-2-induced proliferation and STAT3 and STAT5 phosphorylation in T cells. High affinity IL-2R expression was required for the suppression of IL-2 responses as a novel CD25(-) T cell line proliferated and phosphorylated STAT3 when cultured with tumor cells or rTGFbeta1. Activating T cells with IL-15, which does not use the high affinity IL-2R, completely restored the ability of T cells to phosphorylate STAT3 and STAT5 when cultured with tumor cells. IL-15-treated T cells proliferated normally when cocultured with tumor cells or rTGFbeta1, whereas IL-2 responses were consistently inhibited. Preincubation with IL-15 also restored the ability of T cells to respond to IL-2 by phosphorylating STAT3 and STAT5, and proliferating normally in the presence of tumor cells. IL-2 pretreatment did not restore T cell function. IL-15 also restored T cell responses by T cells from multiple myeloma patients, and against freshly isolated bone marrow tumor samples. Thus, activation of T cells by IL-15 renders T cells resistant to suppression by TGFbeta1-producing tumor cells and rTGFbeta1. This finding may be exploited in the design of new immunotherapy approaches that will rely on T cells avoiding tumor-induced suppression.     

Suppression of DNA-PKcs enhances FGF-2 dependent human endothelial cell proliferation via negative regulation of Akt

Angiogenesis initiation is crucially dependent on endothelial proliferation and can be stimulated by the fibroblast growth factor 2 (FGF-2). The DNA dependent protein kinase (DNA-PK), long known for its importance in repairing DNA double strand breaks, belongs to the phosphatidylinositol-3 kinase (PI3-K) super family and has recently been identified as one of the enzymes phosphorylating and activating Akt. Due to its similarity with PI3-K, we hypothesized that DNA-PK may have similar effects on endothelial angiogenic processes and signalling. We used primary endothelial cells (HUVEC and PAEC) and human microvascular endothelial cells (HMEC) to study the role of DNA-PK in endothelial proliferation and signalling. DNA-PKcs suppression with the compound NU7026 or with siRNA induced basal endothelial cell proliferation as well as enhanced FGF-2 dependent proliferation. This was associated with an increase in phosphorylated Akt. Tube formation was not affected by DNA-PKcs clearly showing that the role of DNA-PK in endothelial processes differs from that of PI3-K. Our findings indicate DNA-PK as an important enzyme maintaining the quiescent endothelial phenotype by actively inhibiting Akt thus restraining endothelial cell proliferation preventing excessive growth.     

Suppression of human monocyte function against Candida albicans by autologous IL-2-induced lymphokine-activated killer cells

We have previously reported that IL-2-induced lymphokine-activated killer (LAK) cells have the capacity to lyse autologous and allogeneic monocytes. To understand the biologic significance of this interaction, we investigated the function of human monocytes against the opportunistic pathogen, Candida albicans, subsequent to a short exposure to autologous LAK cells. A highly sensitive radiolabel assay, which makes use of the incorporation of [3H]glucose into residual Candida after their incubation with monocytes, was developed to measure antifungal activity. Cultured monocytes, after 2 to 6 h exposure to LAK cells, were found to be substantially suppressed in their ability to control fungal growth. Moreover, monocytes cultured in the presence of granulocyte/macrophage (GM)-CSF or IL-3, were even more suppressed in function after a short incubation with LAK cells. The effect of GM-CSF was both time and dose dependent, with peak susceptibility induced after 4 days of culture with as little as 10 U/ml of the cytokine. These GM-CSF-cultured monocytes, however, were relatively resistant to inhibition by freshly isolated large granular lymphocytic NK cells. Therefore, IL-2 induces in large granular lymphocytic cells the capacity to inhibit monocyte function. In contrast to GM-CSF and IL-3, IFN-gamma was found to have a protective effect on monocytes, because monocytes cultured 4 days in IFN-gamma were not significantly inhibited by LAK cells. These results indicate that LAK cells may be involved in regulation of monocyte function and suggest that the state of differentiation induced by different cytokines may dictate the level of control of the monocytes by LAK cells.     

Modulation of IL-2-induced human B cell proliferation in the presence of human 50-kDa B cell growth factor and IL-4

Several human T cell derived factors capable of stimulating human B cells to synthesize DNA have been previously described. One such factor exhibits an apparent m.w. of 50,000 Da and has been termed 50-kDa-B cell growth factor (BCGF). In this report, we show that a human B cell proliferation pathway based on the sequential action of anti-mu antibody, 50-kDa-BCGF and IL-2 is inhibited in the presence of human rIL-4. Although IL-4 itself is capable of triggering B cell DNA synthesis as measured at 72 h, this lymphokine inhibits, in a dose-related manner, the 50-kDa-BCGF driven response of B cells to IL-2 when such proliferation is determined after 144 h. This inhibition takes place at an early step of the B cell activation and does not require the presence of IL-4 during the whole culture period. Such inhibitory activity of IL-4 is specific to the IL-2-induced B cell proliferation because DNA synthesis measured in the presence of semi-purified human 12-kDa-BCGF is not affected by the presence of IL-4. Our results suggest that a particular pathway of human B cell activation leading to the proliferation of these cells in the presence of IL-2 could be either up- or down-modulated by 50-kDa-BCGF and IL-4, respectively.     

TGF-beta2 inhibits AKT activation and FGF-2-induced corneal endothelial cell proliferation

The corneal endothelial cells form a boundary layer between anterior chamber and cornea. This single cell layer is important to maintain cornea transparency by eliciting net fluid transport into the anterior chamber. Injuries of the corneal endothelial layer in humans lead to corneal swelling and translucence. This hindrance is thought to be due to limited proliferative capacity of the endothelial layer. Fibroblast growth factor 2 (FGF-2) and transforming growth factor-beta 2 (TGF-beta2) are both found in aqueous humor, and these two cytokines promote and inhibit cell growth, respectively. The intracellular signaling mechanisms by which TGF-beta2 suppresses the mitogenic response to FGF-2, however, remain unclear. We have addressed this question by investigating potential crosstalk between FGF-2-induced and TGF-beta2-regulated intracellular signaling events in cultured bovine corneal endothelial (BCE) cells. We found that TGF-beta2 and FGF-2 oppositely affect BCE cell proliferation and TGF-beta2 can override the stimulating effects of FGF-2 by increasing COX-2 expression in these cells. Consistent with these findings, overexpression of COX-2 significantly reduced FGF-2-induced cell proliferation whereas a COX-2 specific inhibitor NS398 reversed the effect of TGF-beta2 on FGF-2-induced cell proliferation. The COX-2 product prostaglandin E2 (PGE-2) blocks FGF-2-induced cell proliferation. Whereas FGF-2 stimulates cell proliferation by activating the AKT pathway, TGF-beta2 and PGE-2 both inhibit this pathway. In accordance with the effect of PGE-2, cAMP also inhibits FGF-2-induced AKT activation. These findings suggest that the mitogenic response to FGF-2 in vivo in the corneal endothelial layer may be inhibited by TGF-beta2-induced suppression of the PI3-kinase/AKT signaling pathway.     

Inhibition by specific monosaccharides of interleukin 2-induced thymocyte proliferation

When rat thymocytes are cultured for 3 days in serum-free medium and are stimulated to divide by interleukin 2 (IL 2), concanavalin A, or sodium periodate oxidation, addition to the medium of 10–25 mMd-ribose, 2-deoxy-d-ribose, or N-acetyl-d-galactosamine inhibits by 40% or more the incorporation of [³H]thymidine. d-ribose and lectin-free IL 2 generated from sodium periodate oxidation of rat spleen cells were used to study the characteristics of this inhibition and to test possible mechanisms of inhibition. Viability of thymocytes cultured with d-ribose is similar to that of cells cultured without this sugar. In order to be inhibitory, d-ribose has to be added to the cultures within the first 24 hr, and the inhibition can be prevented if the sugar is removed 18–24 hr after the start of culture. d-Ribose does not block the absorption of IL 2 by unstimulated rat thymocytes or by concanavalin A-generated thymic or splenic blast cells. When thymocytes are cultured with d-ribose for 24 hr, inactivated with mitomycin C, and then cultured for 3 days with fresh mitogenically stimulated cells, [³H]thymidine incorporation into the latter is not altered. This suggests that the sugar does not generate suppressor cells or suppressor supernates. d-Ribose does not appear to be a general metabolic inhibitor since [³H]leucine incorporation into thymocyte proteins and the release of [³H]leucine into medium after a 2-hr. [³H]leucine pulse are not altered by d-ribose. Trivial or artifactual effects (nonspecific cytotoxicity, changes in thymidine transport, or changes in isotonicity of the culture medium) cannot explain the inhibition. A hypothetical mechanism of inhibition is discussed.     

Exploiting tumor cell senescence in anticancer therapy

Cellular senescence is a physiological process of irreversible cell-cycle arrest that contributes to various physiological and pathological processes of aging. Whereas replicative senescence is associated with telomere attrition after repeated cell division, stress-induced premature senescence occurs in response to aberrant oncogenic signaling, oxidative stress, and DNA damage which is independent of telomere dysfunction. Recent evidence indicates that cellular senescence provides a barrier to tumorigenesis and is a determinant of the outcome of cancer treatment. However, the senescence-associated secretory phenotype, which contributes to multiple facets of senescent cancer cells, may influence both cancer-inhibitory and cancer-promoting mechanisms of neighboring cells. Conventional treatments, such as chemo- and radiotherapies, preferentially induce premature senescence instead of apoptosis in the appropriate cellular context. In addition, treatment-induced premature senescence could compensate for resistance to apoptosis via alternative signaling pathways. Therefore, we believe that an intensive effort to understand cancer cell senescence could facilitate the development of novel therapeutic strategies for improving the efficacy of anticancer therapies. This review summarizes the current understanding of molecular mechanisms, functions, and clinical applications of cellular senescence for anticancer therapy. [BMB Reports 2014; 47(2): 51-59]     

Fluvastatin attenuates IGF-1-induced ERK1/2 activation and cell proliferation by mevalonic acid depletion in human mesangial cells

AimsInsulin-like growth factor (IGF)-1 is a major mitogenic growth factor for mesangial cells (MCs). Statins slow the progression of chronic kidney disease by affecting inflammatory cell signaling pathways, in addition to improving lipid profile, however, no studies have investigated the effects of fluvastatin on mitogen-activated protein (MAP) kinase activity or MC proliferation in kidney cells. We investigated the effects of fluvastatin on IGF-1-induced activation of intracellular signal pathways and MC proliferation, and examined the inhibitory mechanisms of fluvastatin.Main methodsWestern blotting and cell proliferation assay were used.Key findingsIGF-1 induced phosphorylation of extracellular-related kinase (ERK)1/2, MAP or ERK kinase (MEK)1/2, and Akt, expression of cyclin D1, and MC proliferation in cultured human MCs. Fluvastatin or PD98059, an MEK1 inhibitor, completely abolished IGF-1-induced MEK1/2 and ERK1/2 phosphorylation and MC proliferation, whereas inhibition of Akt had no effect on MC proliferation. Mevalonic acid prevented fluvastatin inhibition of IGF-1-induced MEK1/2 and ERK1/2 phosphorylation, cyclin D1 expression, and MC proliferation.SignificanceFluvastatin inhibits IGF-1-induced activation of the MAP kinase pathway and MC proliferation by mevalonic acid depletion, and might have renoprotective effects by inhibiting IGF-1-mediated MC proliferation.     

The tumor suppressor PTEN is necessary for human Sprouty 2-mediated inhibition of cell proliferation

Sprouty family proteins are novel regulators of growth factor actions. Human Sprouty 2 (hSPRY2) inhibits the proliferation of a number of different cell types. However, the mechanisms involved in the anti-proliferative actions of hSPRY2 remain to be elucidated. Here we have demonstrated that hSPRY2 increases the amount of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and decreases its phosphorylation. The resultant increase in PTEN activity is reflected in decreased activation of Akt by epidermal growth factor and serum. Consistent with increased PTEN activity, in hSPRY2-expressing cells, the progression of cells from the G1 to S phase is decreased. By using PTEN null primary mouse embryonic fibroblasts and their isogenic controls as well as small interfering RNA against PTEN, we demonstrated that PTEN is necessary for hSPRY2 to inhibit Akt activation by epidermal growth factor as well as cell proliferation. Overall, we concluded that hSPRY2 mediates its anti-proliferative actions by altering PTEN content and activity.     

Suppression of macrophage antimicrobial activity by a tumor cell product

Medium conditioned by tumor cells (TCM) and certain nonmalignant cells contains a trypsin-sensitive factor that suppresses macrophage oxidative metabolism. Because the killing of intracellular pathogens such as Toxoplasma gondii and Leishmania donovani by macrophages is largely oxygen-dependent, we tested the effect of TCM on the antiprotozoal activity of mouse peritoneal macrophages. After 24 hr of cultivation with TCM, in vivo and in vitro activated macrophages could no longer kill toxoplasmas or inhibit their replication. In vivo administration of TCM resulted in similar impairment. The leishmanicidal activity of resident and activated macrophages, when measured 6 hr after infection, was markedly suppressed by in vitro exposure to TCM. The addition of exogenous H2O2 in the form of glucose-glucose oxidase reconstituted the capacity of TCM-exposed macrophages to kill L. donovani promastigotes as quickly as control cells. Thus, TCM appears to deactivate macrophages by the functional criteria of suppressed antitoxoplasmal and antileishmanial activity, as well as by the biochemical criterion of suppressed oxidative metabolism.     

MAPK15 mediates BCR-ABL1-induced autophagy and regulates oncogene-dependent cell proliferation and tumor formation

A reciprocal translocation of the ABL1 gene to the BCR gene results in the expression of the oncogenic BCR-ABL1 fusion protein, which characterizes human chronic myeloid leukemia (CML), a myeloproliferative disorder considered invariably fatal until the introduction of the imatinib family of tyrosine kinase inhibitors (TKI). Nonetheless, insensitivity of CML stem cells to TKI treatment and intrinsic or acquired resistance are still frequent causes for disease persistence and blastic phase progression experienced in patients after initial successful therapies. Here, we investigated a possible role for the MAPK15/ERK8 kinase in BCR-ABL1-dependent autophagy, a key process for oncogene-induced leukemogenesis. In this context, we showed the ability of MAPK15 to physically recruit the oncogene to autophagic vesicles, confirming our hypothesis of a biologically relevant role for this MAP kinase in signal transduction by this oncogene. Indeed, by modeling BCR-ABL1 signaling in HeLa cells and taking advantage of a physiologically relevant model for human CML, i.e. K562 cells, we demonstrated that BCR-ABL1-induced autophagy is mediated by MAPK15 through its ability to interact with LC3-family proteins, in a LIR-dependent manner. Interestingly, we were also able to interfere with BCR-ABL1-induced autophagy by a pharmacological approach aimed at inhibiting MAPK15, opening the possibility of acting on this kinase to affect autophagy and diseases depending on this cellular function. Indeed, to support the feasibility of this approach, we demonstrated that depletion of endogenous MAPK15 expression inhibited BCR-ABL1-dependent cell proliferation, in vitro, and tumor formation, in vivo, therefore providing a novel “druggable” link between BCR-ABL1 and human CML.