Measuring BrdU Incorporation Research Articles

CG '806, a Highly Potent First-in-Class Pan-FLT3/BTK Inhibitor, Targets Multiple Clusters of Kinases Operative in Diverse Subtypes of Acute Myeloid Leukemia

CG'806 is a non-covalent pan-FMS like tyrosine kinase 3 (FLT3)/ Bruton's Tyrosine Kinase (BTK) multi-kinase inhibitor. This small molecule was designed to inhibit the wild type (WT) and C481S mutant forms of BTK, as well as the WT and mutant forms (including the internal tandem duplication, ITD) of the FLT3 receptor tyrosine kinase. Previously (2017 AACR Hematologic Malignancies abstract # 25 and #44), CG'806 was shown to have nM potency against FLT3 mutation-driven (including ITD and point mutations in tyrosine kinase domain) human acute myeloid leukemia (AML) primary patient cells, and in cultured human (eg. MV4-11 and MOLM-13) and mouse (Ba/F3) AML cell lines. CG'806 inhibited FLT3 signaling, induced apoptosis, and eradicated MV4-11 tumors with no observed toxicity in murine xenograft models. Because AML cells demonstrate extreme heterogeneity in gene expression and because FLT3 mutations account for no more than 30% of AML patients, the effect of CG'806 was further evaluated in AML cells without FLT3 mutations. Immunoblotting demonstrated that CG'806 potently decreased phospho-BTK, phospho-aurora kinases A/B/C and phospho-H3S10 without affecting FLT3 activity in AML cells having low/normal expression levels of homozygous WT FLT3 (eg. KG-1 and NOMO-1). Measurement of BrdU incorporation by flow cytometry demonstrated that CG'806 induced G2/M cell cycle arrest or polyploidy and subsequent apoptosis in FLT3 WT AML cell lines. Interestingly, CG'806 was found to induce G0/G1 cell cycle arrest but not polyploidy in FLT3-ITD driven MV4-11 and MOLM-13 cells with no effect on activity of BTK or aurora kinases at concentrations that completely inhibited FLT3 signaling (FLT3/STAT5/ERK). These findings indicate that the molecular mechanism of cell killing by CG'806 is context-dependent and varies in different AML subtypes. Evaluation of enzyme inhibitory activity (IC50) against 583 kinase enzymes, as well as binding affinities (Kd) with 483 kinase proteins, revealed pM to low nM IC50 and Kd values for CG'806 against the FLT3, BTK, AURK, c-Src, c-Met, c-Ret, DDR, TRK and STE/MAPK families of kinases. The ability of CG'806 to target multiple families or clusters of kinases is responsible for its broad efficacy in different AML subtypes driven by different genomic aberrations. Together, these findings illustrate the ability of CG'806 to inhibit entire FLT3 family of kinases, BTK and the AURK families and pathways that are operative in AML, thereby supporting the development of CG'806 for treatment of patients with FLT3-ITD and other subpopulations of patients with AML. DisclosuresZhang:Aptose Biosciences, Inc.: Employment. Local:Aptose Biosciences, Inc.: Employment. Benbatoul:Aptose Biosciences, Inc.: Employment. Folger:Aptose Biosciences, Inc.: Employment. Sheng:Aptose Biosciences, Inc.: Employment. Howell:Aptose Biosciences, Inc.: Membership on an entity's Board of Directors or advisory committees. Rice:Aptose Biosciences, Inc.: Employment, Equity Ownership, Patents & Royalties.

CG '806, a First-in-Class Non-Covalent Pan-FLT3/BTK Inhibitor, Exerts Superior Potency Against B-Cell Malignant Cells

CG'806 is a non-covalent pan-FMS like tyrosine kinase 3 (FLT3)/Bruton's tyrosine kinase (BTK) multi-kinase inhibitor that potently inhibits wild type (WT) and mutant forms of FLT3 and BTK. As described previously (2017 AACR Hematologic Malignancies abstract #25 and #44), CG'806 inhibits on FLT3-ITD-driven acute myeloid leukemia (AML) and other forms of AML. Because CG'806 targets the WT BTK (IC50 = 8 nM) and the C481S-BTK (IC50 = 2.5 nM), which is a mutant form resistant to ibrutinib and other covalent BTK inhibitors, this small molecule was profiled for its actions on various types of B-cell malignancies in which B cell receptor (BCR) and BTK signaling are reported to play pivotal pathogenic roles. Against 10 malignant B-cell lines, while CG'806 reduced the level of BTK-Y223 phosphorylation, it was less potent than ibrutinib. In contrast, CG'806 was 50-6,000 times more potent (lower IC50) in the ability to kill the malignant B cells across all tested cell lines evaluated by MTS-based assays of cell proliferation and inhibition of colony formation. Hence, the efficacy of killing malignant B cells by CG'806 and by ibrutinib did not correlate with the degree of BTK inhibition. Pathway analyses (KinomeScan and immunoblotting) demonstrated that CG'806, in addition to inhibiting intracellular BTK, potently inhibited the activity of aurora kinases A, B and C, phospho-H3S10, FLT3 and ERK that are differentially expressed and operative in various cell lines representing mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), B-acute lymphocytic leukemia (B-ALL) and Burkitt's lymphoma. Indeed, measuring BrdU incorporation and Annexin V by flow cytometry, CG'806 directly induced polyploidy and triggered apoptosis in malignant B cells at picomolar (pM) to low nanomolar (nM) concentrations, while ibrutinib potently inhibited BTK activity but was unable to induce apoptosis at lower than micromolar (µM) concentrations. These data distinguish CG'806 from ibrutinib and other covalent and non-covalent BTK inhibitors, and illustrate that CG'806 has activity across a wide range of malignant B cell lines including those intolerant, refractory or resistant (due to the presence of a BTK-C481S mutation) to ibrutinib and other BTK inhibitors. [Display omitted] DisclosuresZhang:Aptose Biosciences, Inc.: Employment. Local:Aptose Biosciences, Inc.: Employment. Benbatoul:Aptose Biosciences, Inc.: Employment. Folger:Aptose Biosciences, Inc.: Employment. Sheng:Aptose Biosciences, Inc.: Employment. Howell:Aptose Biosciences, Inc.: Membership on an entity's Board of Directors or advisory committees. Rice:Aptose Biosciences, Inc.: Employment, Equity Ownership, Patents & Royalties.

CG '806, a Novel Pan-FLT3/BTK Multi-Kinase Inhibitor, Induces Cell Cycle Arrest, Apoptosis or Autophagy in AML Cells Depending on FLT3 Mutation Status

Gain-of-function mutations of the fms-like tyrosine kinase 3 (FLT3) play a pivotal role in hematopoietic malignancies (Gilliland et al., 2002). Therefore, FLT3 kinase inhibitors are important component in the treatment of acute myeloid leukemia (AML). However, relapse or resistance to these inhibitors are frequent because of secondary acquired mutations in FLT3. Recently, we reported on a novel small molecular multi-kinase inhibitor, CG'806, which showed promising effects in AML harboring FLT3-internal-tandem-deletion (ITD) mutations, tyrosine kinase domain (TKD) mutations, or both by inhibiting FLT3/Bruton's tyrosine kinase (BTK)/aurora kinase (AuroK) activation. We observed an impressive inhibition of leukemic cell proliferation (i.e., IC50s at sub-nanomolar or low nanomolar levels, Zhang, et al., AACR Hematological Malignancies, 2017).To further characterize the mechanisms underlying this anti-leukemia effect, we investigated the impact of CG'806 on cell cycle progression. Measuring BrdU incorporation by flow cytometry, CG'806 triggered profound G1 cell cycle arrest in FLT3-ITD-mutated cells. Interestingly, CG'806 triggered G2/M phase arrest in FLT3 wild type (WT) cells. Immunoblotting demonstrated that the G1 arrest was mediated by downregulation of signaling associated with p-FLT3-ITD, the p-AKT/p-mTOR/cyclin D1/p-Rb axis, and that of cyclin B1/A2, cdk1/cdc2, and cdk4/6 in FLT3-ITD-mutated cells. This was not observed in FLT3 WT cells. In addition, c-Myc, a primary regulator of the G1/S transition, was downregulated in FLT3-mutated but not in WT cells treated with CG'806. In support of this finding, knockdown of c-Myc with siRNA increased the G1-arrested population in FLT3-mutated cells, suggesting a critical role of c-Myc in the CG'806-induced G1 arrest. However, only suppression of p-BTK and p-AuroK but not p-FLT3, and no modulation of G1 arrest-related proteins was observed in FLT3 WT cells.Interestingly, we observed autophagy induction in FLT3 WT cells but induction of apoptosis in FLT3 mutated cells after exposure to low nanomolar CG'806 for 24 hours as evidenced by modulated LC3-II and cleaved-caspase 3 levels. Inhibiting autophagy with 3-methyladenine (3-MA) partially reversed CG'806-induced G2/M arrest in FLT3 WT cells, suggesting that autophagy induction may also be involved in G2/M arrest besides suppression of AuroK and BTK in FLT3 WT cells.Next, we investigated if nucleoside analogues and intercalating agents enhance activity of CG'806, and observed that combination of CG'806 with cytarabine or idarubicin profoundly enhanced pro-apoptotic effects.Conclusions: CG'806 exerts profound suppression of cell proliferation by arresting cell cycle progression at G1 phase in FLT3-mutant AML cells which is associated with inhibition of mutant FLT3 and downstream p-AKT/p-mTOR/cyclin D1/p-Rb axis. CG'806 exerts a G2/M arrest in FLT3 WT cells associated with inhibition of aurora and BTK kinases and induction of autophagy. CG'806 sensitized AML to standard chemotherapeutic agents-mediated cytotoxicity. Taken together, these data support the development of CG'806 for AML patients with FLT3-ITD, FLT3-ITD with additional TKD mutations, as well as FLT3-WT. DisclosuresZhang:Aptose Biosciences, Inc.: Employment. Rice:Aptose Biosciences, Inc.: Employment, Equity Ownership, Patents & Royalties.

Cyclin F Downregulation Affects Epithelial-Mesenchymal Transition Increasing Proliferation and Migration of the A-375 Melanoma Cell Line.

BackgroundCyclins are well-known cell cycle regulators. The activation of cyclin-dependent kinases by cyclins allows orchestration of the complicated cell cycle machinery and drives the cell from the G1 phase to the end of the mitotic phase. In recent years, it has become evident that cyclins are involved in processes beyond the cell cycle. Cyclin F does not activate CDKs but forms part of the Skp1-Cul1-F-box (SCF) complex where it is responsible for protein target recognition and subsequent degradation in a proteasome-dependent manner.ResultsHere, we report that the downregulation of cyclin F in the A-375 melanoma cell line increases cell viability and colony formation in a cell cycle independent manner. Lower levels of cyclin F do not appear to affect the cell cycle, based on flow cytometry measuring BrdU incorporation and propidium iodide staining. By means of immunofluorescence staining and Western blot analysis, we observed changes in cell morphology-related markers which suggested ongoing epithelial-mesenchymal transition (EMT) in response to cyclin F downregulation. Increases in vimentin and N-cadherin protein levels, decreases in levels of epithelial markers such as ZO-1, along with changes in morphology to a spindle-like shape with the appearance of actin stress fibers, are all hallmarks of EMT. These changes are associated with increased invasive and migratory potential, based on 2D migration assays. Moreover, we observe an increase in RhoABC, talin and paxillin levels, the proteins involved in controlling cell signaling and motility. Lastly, upon knocking down cyclin F expression, we observed a decrease in thrombospondin-1 expression, suggesting a role of cyclin F in angiogenesis.ConclusionCyclin F depletion induces proliferation and EMT processes in the A-375 melanoma model.

Metformin Increases Proliferative Activity and Viability of Multipotent Stromal Stem Cells Isolated from Adipose Tissue Derived from Horses with Equine Metabolic Syndrome.

In this study, we investigated the influence of metformin (MF) on proliferation and viability of adipose-derived stromal cells isolated from horses (EqASCs). We determined the effect of metformin on cell metabolism in terms of mitochondrial metabolism and oxidative status. Our purpose was to evaluate the metformin effect on cells derived from healthy horses (EqASCHE) and individuals affected by equine metabolic syndrome (EqASCEMS). The cells were treated with 0.5 μM MF for 72 h. The proliferative activity was evaluated based on the measurement of BrdU incorporation during DNA synthesis, as well as population doubling time rate (PDT) and distribution of EqASCs in the cell cycle. The influence of metformin on EqASC viability was determined in relation to apoptosis profile, mitochondrial membrane potential, oxidative stress markers and BAX/BCL-2 mRNA ratio. Further, we were interested in possibility of metformin affecting the Wnt3a signalling pathway and, thus, we determined mRNA and protein level of WNT3A and β-catenin. Finally, using a two-tailed RT-qPCR method, we investigated the expression of miR-16-5p, miR-21-5p, miR-29a-3p, miR-140-3p and miR-145-5p. Obtained results indicate pro-proliferative and anti-apoptotic effects of metformin on EqASCs. In this study, MF significantly improved proliferation of EqASCs, which manifested in increased synthesis of DNA and lowered PDT value. Additionally, metformin improved metabolism and viability of cells, which correlated with higher mitochondrial membrane potential, reduced apoptosis and increased WNT3A/β-catenin expression. Metformin modulates the miRNA expression differently in EqASCHE and EqASCEMS. Metformin may be used as a preconditioning agent which stimulates proliferative activity and viability of EqASCs.

Inhibition of skin tumor promotion by TPA using a combination of topically applied ursolic acid and curcumin.

Prevention remains an important strategy to reduce the burden of cancer. One approach to prevent cancer is the use of phytochemicals in various combinations as safe and effective cancer preventative agents. The purpose of this study was to examine the effects of the combination of ursolic acid (UA) and curcumin (Curc) for potential combinatorial inhibition of skin tumor promotion using the mouse two-stage skin carcinogenesis model. In short-term experiments, the combination of UA + Curc given topically prior to 12-O-tetradecanoylphorbol-13-acetate (TPA) significantly inhibited activation of epidermal EGFR, p70S6K, NF-κB p50, Src, c-Jun, Rb, and IκBα. Levels of c-Fos, c-Jun, and Cox-2 were also significantly reduced by the combination compared to the TPA treated group. The alterations in these signaling pathways by the combination of UA + Curc were associated with decreased epidermal proliferation as assessed by measuring BrdU incorporation. Significant effects were also seen with the combination on epidermal inflammatory gene expression and dermal inflammation, with the greatest effects on expression of IL-1β, IL-6, IL-22, and CXCL2. Furthermore, results from skin tumor experiments demonstrated that the combination of UA + Curc given topically significantly inhibited mouse skin tumor promotion by TPA to a greater extent than the individual compounds given alone. The greatest effects were seen on tumor free survival, tumor size, and tumor weight, although tumor incidence and multiplicity were also further reduced by the combination. These results demonstrate the potential cancer chemopreventive activity and mechanism(s) for the combination of UA + Curc.

Abstract 278: Inhibition of skin tumor promotion by TPA using a combination of topically applied ursolic acid and curcumin

Abstract Despite the many advances that have been made in understanding cancer development and the discovery of new therapies, cancer still remains the second leading cause of death in the US. In addition, approximately two out of five people are expected to be diagnosed with an invasive cancer in their lifetime. Prevention remains an important strategy to reduce the burden of cancer. One approach to prevent cancer is the use of phytochemicals in various combinations as safe and effective cancer preventive agents. The purpose of this study was to examine the effects of the combination of ursolic acid (UA) and curcumin (Curc) for potential synergistic chemopreventive activity to inhibit tumor promotion using the mouse two-stage skin carcinogenesis model. UA and Curc alone have been shown to inhibit tumorigenesis in several mouse models, and both have been shown to inhibit mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA). The anti-cancer activity of these phytochemicals is due primarily to their anti-inflammatory, anti-oxidant, and anti-proliferative effects. These effects are accomplished by acting on a broad spectrum of signaling pathways. In initial short-term experiments (mice treated twice weekly for two weeks with TPA), the combination of UA and Curc pretreated topically inhibited TPA-induced activation of epidermal NF-κB, Cox-2, epidermal growth factor receptor (EGFR) and Stat3 to a greater extent than either compound alone. Additionally, these compounds increased levels of programmed cell death protein 4 (Pdcd4). The alterations in these signaling pathways by the combination of UA and Curc were associated with decreased epidermal proliferation and skin inflammation as assessed by measuring BrdU incorporation and inflammatory gene expression, respectively. Furthermore, results from skin tumor experiments (mice initiated with DMBA and promoted twice weekly with TPA) indicate that the combination of UA and Curc given topically significantly inhibited mouse skin tumor promotion by TPA to a greater extent than the individual compounds given alone. The greatest effects were seen on tumor free survival and on tumor size. These results suggests the potential for the combination of UA and Curc to be used topically for chemopreventive strategies in non-melanoma skin cancer. Citation Format: Lisa Tremmel, Okkyung Rho, John DiGiovanni. Inhibition of skin tumor promotion by TPA using a combination of topically applied ursolic acid and curcumin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 278.

Abstract 5262: The inhibitory effect of combinations of curcumin, ursolic acid, and metformin on skin tumor promotion by TPA

Abstract The purpose of this study was to examine the effects of combinations of ursolic acid (UA), curcumin (Curc), and metformin (Met) for potential synergistic chemopreventive activity to inhibit tumor promotion using the mouse two-stage skin carcinogenesis model. UA and Curc have been shown to inhibit tumorigenesis in several mouse models, and both have been shown to inhibit mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA). The anti-cancer activity of these phytochemicals is due to their anti-inflammatory, anti-oxidant, and anti-proliferative effects. These effects are accomplished by acting on a broad spectrum of signaling pathways. Met has also been shown to have anti-cancer activity. In retrospective studies, type II diabetes patients taking Met have been shown to have a decreased cancer incidence. Met has also been shown to inhibit mouse skin tumor promotion by TPA. Met activates AMP-activated protein kinase (AMPK), which in turn inhibits mammalian target of rapamycin complex 1 (mTORC1) signaling. mTORC1 is a key kinase involved in the phosphatidylinositol-3 kinase (PI3K)/Akt pathway, one of the most commonly altered pathways in human tumors. In initial short-term experiments (mice treated twice weekly for two weeks with TPA), combinations of UA and Curc pretreated topically inhibited TPA-induced activation of epidermal NF-κB, Cox-2, epidermal growth factor receptor (EGFR) and JNK to a greater extent than either compound alone. Additionally, these compounds increased levels of AMPK and programmed cell death protein 4 (Pdcd4). The alterations in these signaling pathways by the combination of UA and Curc were associated with decreased epidermal proliferation and skin inflammation as assessed by measuring BrdU incorporation and inflammatory gene expression, respectively. Further studies using dietary administration of a combination of UA and Curc, as well as combinations of UA or Curc (in diet) with Met administered in the drinking water were conducted. These combinations produced greater inhibition of TPA-induced activation of epidermal NFκB, Cox-2, and EGFR than the compounds given alone. These combinations also decreased epidermal proliferation, again shown by an inhibition of BrdU incorporation. In addition to these data, the results from ongoing skin tumor experiments (mice initiated with DMBA and promoted twice weekly with TPA) using both topical administration and dietary administration of the various combinations will be presented. The current results indicate that certain combinations of UA, Curc and Met may inhibit mouse skin tumor promotion by TPA to a greater extent than the compounds alone. The ability of the combinations to work when given both topically as well as in the diet suggests the potential for broad applications for chemopreventive strategies. Citation Format: Lisa Tremmel, Okkyung Rho, John DiGiovanni. The inhibitory effect of combinations of curcumin, ursolic acid, and metformin on skin tumor promotion by TPA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5262. doi:10.1158/1538-7445.AM2017-5262

Cyanine TruStain™, an effective blocking buffer to eliminate non-specific cyanine-like dye-mediated monocyte binding

Abstract Cyanine dyes and their analogs are commonly used as acceptors in commercially available antibody-tandem fluorophore conjugates. However, it has been confirmed that the dyes have a tendency to bind monocytes or macrophages nonspecifically, which limits the ability to do multi-color flow cytometric analysis for lower density antigens. We are introducing Cyanine TruStain™ Buffer which eliminates this nonspecific binding. This reagent blocks nonspecific binding of monocytes and macrophages to Cyanine dyes, (PE/Cy7, PE/Cy5, PercpCy5.5, APC/Cy7, APC/Fire™ 750, PE/Dazzle™ 594). It does not affect binding of, e.g., CD64, CD14 and other tested antibodies. Multiple Cyanine dyes can be blocked at once and stability data shows no decrease in signal intensity compared to the conjugate alone. In addition the reagent has no impact on cell viability. Mock sorting experiments were performed with PBMC to determine if the Cyanine TruStain™ Buffer stimulates cells to produce cytokines and chemokines. Our data indicates minimal to no stimulation above conjugated antibody alone for IL-6, IL-8, IL-10 and MCP-1 production. We also tested Cyanine TruStain™ Buffer for its ability to negatively affect PBMC proliferation in response to anti-CD3/anti-CD28 stimulation. We observed minimal to no effect on proliferation by measuring BrdU incorporation. In summary, we are introducing a blocking reagent to eliminate nonspecific binding to Cyanine dyes which shows minimal perturbation of cell functions.

Ghrelin inhibits choroid‐retinal cell migration, proliferation and in vitro angiogenesis, under a high glucose environment

PurposeGhrelin is a peptide expressed in many organs and tissues. Recently, ghrelin has been implicated in the pathophysiology of proliferative retinopathy, although its true involvement remains unclear. The aim of this study is to test the effect of ghrelin in the migration, proliferation, apoptosis and in vitro angiogenesis of primate choroid retinal endothelial cells (RF/6A), cultured under high glucose conditions.MethodsRF/6A cells were incubated for 24 hours with different glucose concentrations (0‐300 mM). Cell migration was assessed using wound‐healing assay. Colorimetric immunoassay was used for the quantification of cell proliferation, based on the measurement of BrdU incorporation. Cell apoptosis was assessed by TUNEL technique. For each glucose concentration, the effect of ghrelin (10‐10 to 10‐5 nM) was determined after 24 hours of incubation. The in vitro angiogenesis was assessed by tube formation assay after exposure to the same glucose concentrations and ghrelin (10‐7 nM) for 4 hours.ResultsGhrelin significantly inhibited RF/6A cell migration at every glucose concentrations, although this effect is more consistent under low glucose environment. Ghrelin, at the concentration of 10‐7 nM, significantly reduces cell proliferation at every glucose concentration. In vitro angiogenesis is decreased by ghrelin under a high glucose environment. No differences on the apoptosis assay were seen.ConclusionsIn conclusion, ghrelin significantly inhibits RF/6A cells migration, proliferation and in vitro angiogenesis, under high glucose environment.

Ghrelin inhibits choroid‐retinal cell migration, proliferation and in vitro angiogenesis, under a high glucose environment

PurposeGhrelin is a peptide expressed in many organs and tissues. Recently, ghrelin has been implicated in the pathophysiology of proliferative retinopathy, although its true involvement remains unclear. The aim of this study is to test the effect of ghrelin in the migration, proliferation, apoptosis and in vitro angiogenesis of primate choroid retinal endothelial cells (RF/6A), cultured under high glucose conditions.MethodsRF/6A cells were incubated for 24 hours with different glucose concentrations (0–300 mM). Cell migration was assessed using wound‐healing assay. Colorimetric immunoassay was used for the quantification of cell proliferation, based on the measurement of BrdU incorporation. Cell apoptosis was assessed by TUNEL technique. For each glucose concentration, the effect of ghrelin (10–10 to 10–5 nM) was determined after 24 hours of incubation. The in vitro angiogenesis was assessed by tube formation assay after exposure to the same glucose concentrations and ghrelin (10–7 nM) for 4 hours.ResultsGhrelin significantly inhibited RF/6A cell migration at every glucose concentrations, although this effect is more consistent under low glucose environment. Ghrelin, at the concentration of 10–7 nM, significantly reduces cell proliferation at every glucose concentration. In vitro angiogenesis is decreased by ghrelin under a high glucose environment. No differences on the apoptosis assay were seen.ConclusionsIn conclusion, ghrelin significantly inhibits RF/6A cells migration, proliferation and in vitro angiogenesis, under high glucose environment.

Small proliferative adipocytes: identification of proliferative cells expressing adipocyte markers [Review

It has been thought that adipocytes lack proliferative ability and do not revert to precursor cells. However, numerous findings that challenge this notion have also been reported. The idea that adipocytes dedifferentiate to fibroblast-like cells with increasing cell number was reported in 1975. This possibility has been ignored despite knowledge gained in the 1990s regarding adipocyte differentiation. Several studies on proliferation and dedifferentiation of adipocytes have been published, most of which were conducted from the perspective of regenerative medicine. However, the concept of proliferation of adipocytes remains unclear. In this study, we postulate a new population of adipocytes, which consist of small sized cells (less than 20 μm in diameter) expressing adipocyte markers, such as adiponectin and peroxisome proliferator-activated receptor γ (PPARγ), but not possessing large lipid droplets. These cells show marked ability to incorporate 5-bromo-2'-deoxyuridine (BrdU), for which reason we termed them "small proliferative adipocytes (SPA)". In addition, SPA are observed in the stromal vascular fraction. Since SPA are morphologically different from mature adipocytes, we regarded them as committed progenitor cells. When proliferation of adipocytes in vivo is assessed by measuring BrdU incorporation and expression levels of proliferating cell nuclear antigen (PCNA) in isolated fractions of adipocytes from adipose tissues, subcutaneous SPA proliferate less actively than visceral SPA. Treatment with pioglitazone increases the number of proliferating SPA in subcutaneous, but not visceral, fat, suggesting that SPA may be important in regulating systemic insulin sensitivity and glucose metabolism.

Angiotensins inhibit cell growth in GH3 lactosomatotroph pituitary tumor cell culture: a possible involvement of the p44/42 and p38 MAPK pathways.

The local renin-angiotensin system is present in the pituitary. We investigated the effects of angiotensins on GH3 lactosomatotroph cells proliferation in vitro and the involvement of p44/42 and p38 MAPK inhibitors in the growth-regulatory effects of angiotensins. Materials and Methods. Cell viability using the Mosmann method and proliferation by the measurement of BrdU incorporation during DNA synthesis were estimated. Results. Ang II and ang IV decreased the viability and proliferation of GH3 cells. Inhibitor of p44/42 MAPK attenuated the effects of ang II on cell viability and proliferation but did not affect the ang 5-8-dependent actions. Inhibitor of p38 MAPK prevented the decrease in the number of GH3 cells in ang-II- and ang-IV-treated groups. Conclusions. The growth-inhibitory effect of ang II is possibly mediated by the p44/42 MAPK. The p38 MAPK appears to mediate the inhibitory effects of both ang II and ang 5–8 upon cell survival.

PTTG1 Attenuates Drug-Induced Cellular Senescence

As PTTG1 (pituitary tumor transforming gene) abundance correlates with adverse outcomes in cancer treatment, we determined mechanisms underlying this observation by assessing the role of PTTG1 in regulating cell response to anti-neoplastic drugs. HCT116 cells devoid of PTTG1 (PTTG1−/−) exhibited enhanced drug sensitivity as assessed by measuring BrdU incorporation in vitro. Apoptosis, mitosis catastrophe or DNA damage were not detected, but features of senescence were observed using low doses of doxorubicin and TSA. The number of drug-induced PTTG1−/− senescent cells increased ∼4 fold as compared to WT PTTG1-replete cells (p<0.001). p21, an important regulator of cell senescence, was induced ∼3 fold in HCT116 PTTG1−/− cells upon doxorubicin or Trichostatin A treatment. Binding of Sp1, p53 and p300 to the p21 promoter was enhanced in PTTG1−/− cells after treatment, suggesting transcriptional regulation of p21. p21 knock down abrogated the observed senescent effects of these drugs, indicating that PTTG1 likely suppresses p21 to regulate drug-induced senescence. PTTG1 also regulated SW620 colon cancer cells response to doxorubicin and TSA mediated by p21. Subcutaneously xenografted PTTG1−/− HCT116 cells developed smaller tumors and exhibited enhanced responses to doxorubicin. PTTG1−/− tumor tissue derived from excised tumors exhibited increased doxorubicin-induced senescence. As senescence is a determinant of cell responses to anti-neoplastic treatments, these findings suggest PTTG1 as a tumor cell marker to predict anti-neoplastic treatment outcomes.

Effect of probiotic treatment on the transition from chronic colonic inflammation to dysplasia and cancer.

385 Background: Effective prevention of colitis-associated cancer still needs to be achieved. There is evidence that the microflora plays an important role in the progression of chronic inflammation to neoplastic transformation, so the aim of this study was to evaluate the potential efficacy of the probiotic VSL#3 in preventing this transition. Methods: Chronic colitis was induced in male Sprague Dawley rats by the administration of trinitrobenzene sulfonic acid (TNBS; 30 mg in 50% ethanol ic), followed six weeks later by reactivation with TNBS (5 mg/kg iv) for 3 days. To induce colitis-associated dysplasia and cancer the animals then received TNBS (iv) twice a week for ten weeks. One group of rats received the probiotic in drinking water (VSL#3; ∼5 billion cfu /100 gram body weight) from 1 week prior to initial colitis induction until the time of sacrifice (n = 22-23/group). After sacrifice the colons were removed and analyzed for total macroscopic damage, microscopic damage and presence of dysplasia or cancer. Tissue and serum samples were analyzed for cell proliferation and apoptosis, angiogenic factors and presence of alkaline sphingomyelinase. Results: Those animals receiving the probiotic had significantly less macroscopic and microscopic damage than the vehicle treated-controls in all areas of the colon (p &lt; 0.05). Pathological analysis revealed that 29% of the vehicle treated animals developed carcinoma, which was confined to the proximal region. None of the probiotic treated animals developed carcinoma. Treatment with the probiotic caused a significant shift to a less severe diagnosis with no high-grade dysplasia found in either the proximal or midcolon. Treatment with the probiotic reduced crypt cell proliferation by 22% as assessed by measurement of BrdU incorporation in comparison with the vehicle treated group. Levels of the antiangiogenic factor angiostatin were higher in the probiotic treated animals (5-fold), as were serum levels of alkaline sphingomyelinase (2-fold). Conclusions: Treatment with the probiotic VSL#3 can delay the transition of chronic inflammation to dysplasia and cancer via effects on processes such as modulation of cell proliferation and angiogenesis. No significant financial relationships to disclose.

Combinatory effects of PBDEs and 17β-estradiol on MCF-7 cell proliferation and apoptosis

In the present work, we analyzed whether polybrominated diphenyl ethers (PBDEs) (47,99,100 and 209) interfere with the effect of 17β-estradiol on the proliferation and apoptosis of the MCF-7 cell line. MCF-7 cells were cultured in DMEM without phenol red supplemented with 5% charcoal-treated fetal bovine serum for 3 days with 10nM 17β-estradiol; with 0.1µM, 0.5µM or 1µM of the tested PBDE congeners; or with both 17β-estradiol and a congener. Cell proliferation was determined by measuring BrdU incorporation, and cell apoptosis was measured by caspase-9 activity.No PBDE congener had an effect on basal cell proliferation, but they all significantly decreased basal caspase-9 activity. An additive anti-apoptotic activity and ability to induce cell proliferation was observed in the presence of 17β-estradiol.

All-trans retinoic acid diminishes collagen production in a hepatic stellate cell line via suppression of active protein-1 and c-Jun N-terminal kinase signal

Following acute and chronic liver injury, hepatic stellate cells (HSCs) become activated to undergo a phenotypic transformation into myofibroblast-like cells and lose their retinol content, but the mechanisms of retinoid loss and its potential roles in HSCs activation and liver fibrosis are not understood. The influence of retinoids on HSCs and hepatic fibrosis remains controversial. The purpose of this study was to evaluate the effects of all-trans retinoid acid (ATRA) on cell proliferation, mRNA expression of collagen genes [procollagen α1 (I), procollagen α1 (III)], profibrogenic genes (TGF-β(1), CTGF, MMP-2, TIMP-1, TIMP-2, PAI-1), fibrolytic genes (MMP-3, MMP-13) and the upstream element (JNK and AP-1) in the rat hepatic stellate cell line (CFSC-2G). Cell proliferation was evaluated by measuring BrdU incorporation. The mRNA expression levels of collagen genes [procollagen α1 (I), procollagen α1 (III)], profibrogenic genes (TGF-β(1), CTGF, MMP-2, TIMP-1, TIMP-2, PAI-1), and fibrolytic genes (MMP-3, MMP-13) were quantitatively detected by using real-time PCR. The mRNA expression of JNK and AP-1 was quantified by RT-PCR. The results showed that ATRA inhibited HSCs proliferation and diminished the mRNA expression of collagen genes [procollagen α1 (I), procollagen α1 (III)] and profibrogenic genes (TGF-β(1), CTGF, MMP-2, TIMP-1, TIMP-2, PAI-1), and significantly stimulated the mRNA expression of MMP-3 and MMP-13 in HSCs by suppressing the mRNA expression of JNK and AP-1. These findings suggested that ATRA could inhibit proliferation and collagen production of HSCs via the suppression of active protein-1 and c-Jun N-terminal kinase signal, then decrease the mRNAs expression of profibrogenic genes (TGF-β(1), CTGF, MMP-2, TIMP-1, TIMP-2, PAI-1), and significantly induce the mRNA expression of MMP-3 and MMP-13.

The EphB4 receptor promotes the growth of melanoma cells expressing the ephrin‐B2 ligand

The EphB4 receptor promotes the growth of melanoma cells expressing the ephrin‐B2 ligand

Substance P stimulates bone marrow stromal cell osteogenic activity, osteoclast differentiation, and resorption activity in vitro

IntroductionSP is a neuropeptide distributed in the sensory nerve fibers that innervate the medullar tissues of bone, as well as the periosteum. Previously we demonstrated that inhibition of neuropeptide signaling after capsaicin treatment resulted in a loss of bone mass and we hypothesized that SP contributes to bone integrity by stimulating osteogenesis. Materials and methodsOsteoblast precursors (bone marrow stromal cells, BMSCs) and osteoclast precursors (bone marrow macrophages, BMMs) derived from C57BL/6 mice were cultured. Expression of the SP receptor (NK1) was detected by using immunocytochemical staining and PCR. Effects of SP on proliferation and differentiation of BMSCs were studied by measuring BrdU incorporation, gene expression, alkaline phosphatase activity, and osteocalcin and Runx2 protein levels with EIA and western blot assays, respectively. Effects of SP on BMMs were determined using a BrdU assay, counting multinucleated cells staining positive for tartrate-resistant acid phosphatase (TRAP+), measuring pit erosion area, and evaluating RANKL protein production and NF-κB activity with ELISA and western blot. ResultsThe NK1 receptor was expressed in both BMSCs and BMMs. SP stimulated the proliferation of BMSCs in a concentration-dependent manner. Low concentrations (10−12 M) of SP stimulated alkaline phosphatase and osteocalcin expression, increased alkaline phosphatase activity, and up-regulated Runx2 protein levels, and higher concentrations of SP (10−8 M) enhanced mineralization in differentiated BMSCs. SP also stimulated BMSCs to produce RANKL, but at concentrations too low to evoke osteoclastogenesis in co-culture with macrophages in the presence of SP. SP also activated NF-κB in BMMs and directly facilitate RANKL-induced macrophage osteoclastogenesis and bone resorption activity. ConclusionsNK1 receptors are expressed by osteoblast and osteoclast precursors and SP stimulates osteoblast and osteoclast differentiation and function in vitro. SP neurotransmitter release from sensory neurons could potentially regulate local bone turnover in vivo.

Inhibition of proliferation, VEGF secretion of human neuroendocrine tumor cell line NCI-H727 by an antagonist of growth hormone-releasing hormone (GH-RH) in vitro

Growth hormone-releasing hormone (GH-RH) can stimulate not only growth hormone (GH) secretion by anterior pituitary gland but also proliferation of many cancer cell lines in vitro and in xenografts tumor models in vivo. Several antagonists of GH-RH have been shown to inhibit several cancer growths, but the role of GH-RH antagonists in the regulation of neuroendocrine cancers cell proliferation and tumor progression remains obscure. The aim of the study was to evaluate the influence of JV-1-36 (synthetic GH-RH antagonist) on proliferation and VEGF secretion by human neuroendocrine lung non-small cell carcinoma (NCI-H727) using cell culture model. The in vitro effect of JV-1-36 on the proliferation of NCI-H727 cells was assessed by the measurement of BrdU incorporation by colorimetric immunoassay. The presence of VEGF and membrane GH-RH receptors on the surface of H727 cells were visualized by immunocytochemistry using specific anti-GH-RH receptor antibody directed to the carboxy-terminal region. VEGF secretion to the cell cultures supernatants was assessed by ELISA methods. Immunoreactive cell membrane GH-RH receptors and VEGF–immunopositive cytoplasmatic granules were clearly confined on the surface of nearly all cancer cells. JV-1-36 at the concentration of 10 −6–10 −10 M significantly inhibited growth of H727 cells, compared with untreated controls. In H727 cells, the antiproliferative JV-1-36 effect was associated with a dose-dependent reduction of VEGF secretion. In conclusion, our findings demonstrate the strong evidence for the antiproliferative action of GH-RH antagonist JV-1-36 for the NCI-H727 cells. In addition the suppression of VEGF secretion by H727 cells might contribute, at least in part, to the antitumor action of GH-RH antagonists.