A naringenin-tamoxifen combination impairs cell proliferation and survival of MCF-7 breast cancer cells.

Since over 60% of breast cancers are estrogen receptor positive (ER+), many therapies have targeted the ER. The ER is activated by both estrogen binding and phosphorylation. While anti-estrogen therapies, such as tamoxifen (Tam) have been successful they do not target the growth factor promoting phosphorylation of the ER. Other proliferation pathways such as the phosphatidylinositol-3 kinase, (PI3K) and the mitogen-activated protein kinase (MAPK) pathways are activated in breast cancer cells and are associated with poor prognosis. Thus targeting multiple cellular proliferation and survival pathways at the onset of treatment is critical for the development of more effective therapies. The grapefruit flavanone naringenin (Nar) is an inhibitor of both the PI3K and MAPK pathways. Previous studies examining either Nar or Tam used charcoal-stripped serum which removed estrogen as well as other factors. We wanted to use serum containing medium in order to retain all the potential inducers of cell proliferation so as not to exclude any targets of Nar. Here we show that a Nar-Tam combination is more effective than either Tam alone or Nar alone in MCF-7 breast cancer cells. We demonstrate that a Nar-Tam combination impaired cellular proliferation and viability to a greater extent than either component alone in MCF-7 cells. Furthermore, the use of a Nar-Tam combination requires lower concentrations of both compounds to achieve the same effects on proliferation and viability. Nar may function by inhibiting both PI3K and MAPK pathways as well as localizing ERα to the cytoplasm in MCF-7 cells. Our results demonstrate that a Nar-Tam combination induces apoptosis and impairs proliferation signaling to a greater extent than either compound alone. These studies provide critical information for understanding the molecular mechanisms involved in cell proliferation and apoptosis in breast cancer cells.

Regulation of myosin IIA and filamentous actin during insulin-stimulated glucose uptake in 3T3-L1 adipocytes.

Insulin stimulated glucose uptake requires the colocalization of myosin IIA (MyoIIA) and the insulin-responsive glucose transporter 4 (GLUT4) at the plasma membrane for proper GLUT4 fusion. MyoIIA facilitates filamentous actin (F-actin) reorganization in various cell types. In adipocytes F-actin reorganization is required for insulin-stimulated glucose uptake. What is not known is whether MyoIIA interacts with F-actin to regulate insulin-induced GLUT4 fusion at the plasma membrane. To elucidate the relationship between MyoIIA and F-actin, we examined the colocalization of MyoIIA and F-actin at the plasma membrane upon insulin stimulation as well as the regulation of this interaction. Our findings demonstrated that MyoIIA and F-actin colocalized at the site of GLUT4 fusion with the plasma membrane upon insulin stimulation. Furthermore, inhibition of MyoII with blebbistatin impaired F-actin localization at the plasma membrane. Next we examined the regulatory role of calcium in MyoIIA-F-actin colocalization. Reduced calcium or calmodulin levels decreased colocalization of MyoIIA and F-actin at the plasma membrane. While calcium alone can translocate MyoIIA it did not stimulate F-actin accumulation at the plasma membrane. Taken together, we established that while MyoIIA activity is required for F-actin localization at the plasma membrane, it alone is insufficient to localize F-actin to the plasma membrane.

Dual role for myosin II in GLUT4-mediated glucose uptake in 3T3-L1 adipocytes.

Insulin-stimulated glucose uptake requires the activation of several signaling pathways to mediate the translocation and fusion of GLUT4 vesicles to the plasma membrane. Our previous studies demonstrated that GLUT4-mediated glucose uptake is a myosin II-dependent process in adipocytes. The experiments described in this report are the first to show a dual role for the myosin IIA isoform specifically in regulating insulin-stimulated glucose uptake in adipocytes. We demonstrate that inhibition of MLCK but not RhoK results in impaired insulin-stimulated glucose uptake. Furthermore, our studies show that insulin specifically stimulates the phosphorylation of the RLC associated with the myosin IIA isoform via MLCK. In time course experiments, we determined that GLUT4 translocates to the plasma membrane prior to myosin IIA recruitment. We further show that recruitment of myosin IIA to the plasma membrane requires that myosin IIA be activated via phosphorylation of the RLC by MLCK. Our findings also reveal that myosin II is required for proper GLUT4-vesicle fusion at the plasma membrane. We show that once at the plasma membrane, myosin II is involved in regulating the intrinsic activity of GLUT4 after insulin stimulation. Collectively, our results are the first to reveal that myosin IIA plays a critical role in mediating insulin-stimulated glucose uptake in 3T3-LI adipocytes, via both GLUT4 vesicle fusion at the plasma membrane and GLUT4 activity.

Inhibition of the MAPK pathway alone is insufficient to account for all of the cytotoxic effects of naringenin in MCF-7 breast cancer cells.

Estrogen receptor (ER) antagonists such as tamoxifen (Tam) have been used successfully to treat ER+ breast cancers for more than 30 years. Unfortunately, long term use of Tam can result in resistance. Tam resistance is associated with the activation of growth factor signaling pathways that promote cell proliferation and survival. The mitogen-activated protein kinase (MAPK), is up-regulated in Tam resistant (Tam-R) cells. Previous studies have reported that the flavanone, naringenin (Nar) can inhibit cell proliferation and induce apoptosis in ER+ breast cancer cells. Furthermore, Nar has been shown to inhibit the MAPK signaling pathways in MCF-7 cells. In this report we investigated whether inhibition of MAPK alone is mediating the effects of Nar on cell proliferation and viability. These studies will determine the mechanism of action of Nar. Tam-R MCF-7 breast cancer cells were treated with Nar or U0126, a MAPK kinase inhibitor. Our studies show that while both U0126 and Nar impaired cell proliferation and viability the combination of U0126 and Nar resulted in greater inhibition of cell viability than either compound alone. It has been previously reported that Nar can bind the ER. Our lab has also shown that Nar localizes ERα to a peri-nuclear region of the cell. Confocal microscopy revealed that in U0126 treated cells ERα displayed an even distribution across the cytoplasm as seen in untreated Tam-R cells. These studies suggest that MAPK is not the only target of Nar.

GLUT4 expression in 3T3-L1 adipocytes is repressed by proteasome inhibition, but not by inhibition of calpains.

Because of recent studies showing linkage of type 2 diabetes with the calpain 10 gene, we investigated the ability of calpains to regulate GLUT4 expression in 3T3-L1 adipocytes. Treatment of 3T3-L1 adipocytes with the calpain inhibitor ALLN significantly decreased the mRNA and protein expression of GLUT4. GLUT4 expression was not affected by treatment with the more selective calpain inhibitors PD150606, calpeptin, or a calpastatin peptide. In contrast, treatment with the proteasome inhibitors lactacystin or MG132 repressed GLUT4 mRNA level to 35% (10 microM lactacystin) and 12% (10 microM MG132) of control levels. Therefore, the expression of GLUT4 in 3T3-L1 adipocytes was repressed by proteasome inhibition, but not by inhibition of calpains; the effect of ALLN was due to its ability to inhibit proteasome function, rather than its action to inhibit calpains. Concomitant with the repression of GLUT4 mRNA levels, proteasome inhibition decreased GLUT4 protein levels in 3T3-L1 adipocytes. The decrease in GLUT4 expression occurred at the transcriptional level, as treatment with proteasome inhibitors decreased GLUT4 transcription measured by a nuclear run-on assay. Thus, these data demonstrate a new pathway for the regulation of GLUT4 expression that involves proteasomal degradation of factors that regulate GLUT4 expression.

Calpain facilitates GLUT4 vesicle translocation during insulin-stimulated glucose uptake in adipocytes.

Calpains are a family of non-lysosomal cysteine proteases. Recent studies have identified a member of the calpain family of proteases, calpain 10, as a putative diabetes-susceptibility gene that may be involved in the development of type 2 diabetes. Inhibition of calpain activity has been shown to reduce insulin-stimulated glucose uptake in isolated rat-muscle strips and adipocytes. In this report, we examine the mechanism by which calpain affects insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Inhibition of calpain activity resulted in approx. a 60% decrease in insulin-stimulated glucose uptake. Furthermore, inhibition of calpain activity prevented the translocation of insulin-responsive glucose transporter 4 (GLUT4) vesicles to the plasma membrane, as demonstrated by fluorescent microscopy of whole cells and isolated plasma membranes; it did not, however, alter the total GLUT4 protein content. While inhibition of calpain did not affect the insulin-mediated proximal steps of the phosphoinositide 3-kinase pathway, it did prevent the insulin-stimulated cortical actin reorganization required for GLUT4 translocation. Specific inhibition of calpain 10 by antisense expression reduced insulin-stimulated GLUT4 translocation and actin reorganization. Based on these findings, we propose a role for calpain in the actin reorganization required for insulin-stimulated GLUT4 translocation to the plasma membrane in 3T3-L1 adipocytes. These studies identify calpain as a novel factor involved in GLUT4 vesicle trafficking and suggest a link between calpain activity and the development of type 2 diabetes.

Regulation of myosin light chain kinase during insulin-stimulated glucose uptake in 3T3-L1 adipocytes.

Myosin II (MyoII) is required for insulin-responsive glucose transporter 4 (GLUT4)-mediated glucose uptake in 3T3-L1 adipocytes. Our previous studies have shown that insulin signaling stimulates phosphorylation of the regulatory light chain (RLC) of MyoIIA via myosin light chain kinase (MLCK). The experiments described here delineate upstream regulators of MLCK during insulin-stimulated glucose uptake. Since 3T3-L1 adipocytes express two MyoII isoforms, we wanted to determine which isoform was required for insulin-stimulated glucose uptake. Using a siRNA approach, we demonstrate that a 60% decrease in MyoIIA protein expression resulted in a 40% inhibition of insulin-stimulated glucose uptake. We also show that insulin signaling stimulates the phosphorylation of MLCK. We further show that MLCK can be activated by calcium as well as signaling pathways. We demonstrate that adipocytes treated with the calcium chelating agent, 1,2-b (iso-aminophenoxy) ethane-N,N,N',N'-tetra acetic acid, (BAPTA) (in the presence of insulin) impaired the insulin-induced phosphorylation of MLCK by 52% and the RLC of MyoIIA by 45% as well as impairing the recruitment of MyoIIA to the plasma membrane when compared to cells treated with insulin alone. We further show that the calcium ionophore, A23187 alone stimulated the phosphorylation of MLCK and the RLC associated with MyoIIA to the same extent as insulin. To identify signaling pathways that might regulate MLCK, we examined ERK and CaMKII. Inhibition of ERK2 impaired phosphorylation of MLCK and insulin-stimulated glucose uptake. In contrast, while inhibition of CaMKII did inhibit phosphorylation of the RLC associated with MyoIIA, inhibition of CAMKIIδ did not impair MLCK phosphorylation or translocation to the plasma membrane or glucose uptake. Collectively, our results are the first to delineate a role for calcium and ERK in the activation of MLCK and thus MyoIIA during insulin-stimulated glucose uptake in 3T3-L1 adipocytes.

Calpain facilitates actin reorganization during glucose-stimulated insulin secretion.

Calpain-10 (CAPN10) has been identified as a diabetes susceptibility gene. Previous studies have shown that alterations in calpain activity alter both glucose uptake and insulin secretion. In this report, we investigated the role of calpain activity in the actin reorganization required for glucose-stimulated insulin secretion. In pancreatic INS-1 cells, acute exposure to a high glucose environment stimulated CAPN10 gene expression with a concomitant increase in calpain activity. However, high glucose did not significantly alter expression of the two major ubiquitously expressed calpain family members, CAPN1 and CAPN2. Furthermore, glucose stimulation resulted in the reorganization of actin and inhibition of calpain activity impaired this reorganization in INS-1 cells. Finally, we identified a 54 kDa isoform as the major CAPN10 isoform that associates with the actin cytoskeleton. Based on our findings, we propose that calpain plays a role in facilitating the actin reorganization required for glucose-stimulated insulin secretion in INS-1 cells.

A novel role for myosin II in insulin-stimulated glucose uptake in 3T3-L1 adipocytes.

Insulin-stimulated glucose uptake requires the activation of several signaling pathways to mediate the translocation and fusion of GLUT4 vesicles from an intracellular pool to the plasma membrane. The studies presented here show that inhibition of myosin II activity impairs GLUT4-mediated glucose uptake but not GLUT4 translocation to the plasma membrane. We also show that adipocytes express both myosin IIA and IIB isoforms, and that myosin IIA is recruited to the plasma membrane upon insulin stimulation. Taken together, the data presented here represent the first demonstration that GLUT4-mediated glucose uptake is a myosin II-dependent process in adipocytes. Based on our findings, we hypothesize that myosin II is activated upon insulin stimulation and recruited to the cell cortex to facilitate GLUT4 fusion with the plasma membrane. The identification of myosin II as a key component of GLUT4-mediated glucose uptake represents an important advance in our understanding of the mechanisms regulating glucose homeostasis.

Naringenin inhibits glucose uptake in MCF-7 breast cancer cells: a mechanism for impaired cellular proliferation.

Certain flavonoids inhibit glucose uptake in cultured cells. In this report, we show that the grapefruit flava-none naringenin inhibited insulin-stimulated glucose uptake in proliferating and growth-arrested MCF-7 breast cancer cells. Our findings indicate that naringenin inhibits the activity of phosphoinositide 3-kinase (PI3K), a key regulator of insulin-induced GLUT4 translocation, as shown by impaired phosphorylation of the downstream signaling molecule Akt. Naringenin also inhibited the phosphorylation of p44/p42 mitogen-activated protein kinase (MAPK). Inhibition of the MAPK pathway with PD98059, a MAPK kinase inhibitor, reduced insulin-stimulated glucose uptake by approximately 60%. The MAPK pathway therefore appears to contribute significantly to insulin-stimulated glucose uptake in breast cancer cells. Importantly, decreasing the availability of glucose by lowering the glucose concentration of the culture medium inhibited proliferation, as did treatment with naringenin. Collectively, our findings suggest that naringenin inhibits the proliferation of MCF-7 cells via impaired glucose uptake. Because a physiologically attainable dose of 10 micro M naringenin reduced insulin-stimulated glucose uptake by nearly 25% and also reduced cell proliferation, naringenin may possess therapeutic potential as an anti-proliferative agent.

Naringenin inhibits phosphoinositide 3-kinase activity and glucose uptake in 3T3-L1 adipocytes.

Previous studies have shown that flavonoids inhibit glucose uptake in cultured cells. In this report, we show that the grapefruit flavanone naringenin inhibited insulin-stimulated glucose uptake in 3T3-L1 adipocytes in a dose-dependent manner. Naringenin acts by inhibiting the activity of phosphoinositide 3-kinase (PI3K), a key regulator of insulin-induced GLUT4 translocation. Although naringenin did not alter the phosphotyrosine status of the insulin receptor, insulin receptor substrate proteins, or PI3K, it did inhibit the phosphorylation of the downstream signaling molecule Akt. In an in vitro kinase assay, naringenin inhibited PI3K activity. A physiologically attainable dose of 6 microM naringenin reduced insulin-stimulated glucose uptake by approximately 20%. This inhibitory effect remained 24h after the removal of naringenin from the culture medium. Collectively, our findings suggest that the regular consumption of naringenin in grapefruit may exacerbate insulin resistance in susceptible individuals via impaired glucose uptake in adipose tissue.

MEK inhibitors impair insulin-stimulated glucose uptake in 3T3-L1 adipocytes.

In 3T3-L1 adipocytes, insulin activates three major signaling cascades, the phosphoinositide 3-kinase (PI3K) pathway, the Cbl pathway, and the mitogen-activated protein kinase (MAPK) pathway. Although PI3K and Cbl mediate insulin-stimulated glucose uptake by promoting the translocation of the insulin-responsive glucose transporter (GLUT4) to the plasma membrane, the MAPK pathway does not have an established role in insulin-stimulated glucose uptake. We demonstrate in this report that PI3K inhibitors also inhibit the MAPK pathway. To investigate the role of the MAPK pathway separately from that of the PI3K pathway in insulin-stimulated glucose uptake, we used two specific inhibitors of MAPK kinase (MEK) activity, PD-98059 and U-0126, which reduced insulin-stimulated glucose uptake by approximately 33 and 50%, respectively. Neither MEK inhibitor affected the activation of Akt or PKCzeta/lambda, downstream signaling molecules in the PI3K pathway. Inhibition of MEK with U-0126 did not prevent GLUT4 from translocating to the plasma membrane, nor did it inhibit the subsequent docking and fusion of GLUT4-myc with the plasma membrane. MEK inhibitors affected glucose transport mediated by GLUT4 but not GLUT1. Importantly, the presence of MEK inhibitors only at the time of the transport assay markedly impaired both insulin-stimulated glucose uptake and MAPK signaling. Conversely, removal of MEK inhibitors before the transport assay restored glucose uptake and MAPK signaling. Collectively, our studies suggest a possible role for MEK in the activation of GLUT4.

Genistein inhibits CCAAT/enhancer-binding protein beta (C/EBPbeta) activity and 3T3-L1 adipogenesis by increasing C/EBP homologous protein expression.

The tyrosine kinase inhibitor genistein inhibits 3T3-L1 adipogenesis when present during the first 72 h of differentiation. In this report, we investigated the underlying mechanisms involved in the anti-adipogenic effects of genistein. We found that genistein blocked the DNA binding and transcriptional activity of CCAAT/enhancer-binding protein beta (C/EBPbeta) during differentiation by promoting the expression of C/EBP homologous protein, a dominant-negative member of the C/EBP family. Loss of C/EBPbeta activity was manifested as a loss of differentiation-induced C/EBPalpha and peroxisome-proliferator-activated receptor gamma protein expression and a dramatic reduction in lipid accumulation. Further, we documented for the first time that C/EBPbeta was tyrosine-phosphorylated in vivo during differentiation and in vitro by activated epidermal growth factor receptor. Genistein inhibited both of these events. Collectively, these results indicate that genistein blocks adipogenesis and C/EBPbeta activity by increasing the level of C/EBP homologous protein and possibly by inhibiting the tyrosine phosphorylation of C/EBPbeta.

Rb regulates C/EBPbeta-DNA-binding activity during 3T3-L1 adipogenesis.

Two pathways are initiated upon 3T3-L1 preadipocyte differentiation: the reentry of cells into the cell cycle and the initiation of a cascade of transcriptional events that "prime" the cell for differentiation. The "priming" event involves the synthesis of members of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors. However, the relationship between these two pathways is unknown. Here we report that in the 3T3-L1 preadipocytes induced to differentiate, cell cycle progression and the initiation of differentiation are linked by a cell cycle-dependent Rb-C/EBPbeta interaction. Cell cycle arrest in G1 by l-mimosine inhibited differentiation-induced C/EBPbeta-DNA-binding activity and Rb phosphorylation. However, cell cycle arrest after the G1/S transition by aphidicolin or nocodazole did not prevent C/EBPbeta-DNA-binding activity or Rb phosphorylation. Furthermore, hypophosphorylated Rb and C/EBPbeta coimmunoprecipitated, whereas phosphorylated Rb and C/EBPbeta did not. Electrophoretic mobility shift assays demonstrated that recombinant hypophosphorylated Rb decreased C/EBPbeta-DNA-binding activity and that Rb overexpression inhibited C/EBPbeta-induced transcriptional activation of a C/EBPalpha-promoter-luciferase reporter gene. We conclude that C/EBPbeta-DNA-binding activity is regulated by its interaction with hypophosphorylated Rb, thereby linking the progression of the cell cycle to the initiation of differentiation during 3T3-L1 adipogenesis.

Inhibition of insulin-dependent glucose uptake by trivalent arsenicals: possible mechanism of arsenic-induced diabetes.

Chronic exposures to inorganic arsenic (iAs) have been associated with increased incidence of noninsulin (type-2)-dependent diabetes mellitus. Although mechanisms by which iAs induces diabetes have not been identified, the clinical symptoms of the disease indicate that iAs or its metabolites interfere with insulin-stimulated signal transduction pathway or with critical steps in glucose metabolism. We have examined effects of iAs and methylated arsenicals that contain trivalent or pentavalent arsenic on glucose uptake by 3T3-L1 adipocytes. Treatment with inorganic and methylated pentavalent arsenicals (up to 1 mM) had little or no effect on either basal or insulin-stimulated glucose uptake. In contrast, trivalent arsenicals, arsenite (iAs(III)), methylarsine oxide (MAs(III)O), and iododimethylarsine (DMAs(III)O) inhibited insulin-stimulated glucose uptake in a concentration-dependent manner. Subtoxic concentrations of iAs(III) (20 microM), MAs(III)O (1 microM), or DMAs(III)I (2 microM) decreased insulin-stimulated glucose uptake by 35-45%. Basal glucose uptake was significantly inhibited only by cytotoxic concentrations of iAs(III) or MAs(III)O. Examination of the components of the insulin-stimulated signal transduction pathway showed that all trivalent arsenicals suppressed expression and possibly phosphorylation of protein kinase B (PKB/Akt). The concentration of an insulin-responsive glucose transporter (GLUT4) was significantly lower in the membrane region of 3T3-L1 adipocytes treated with trivalent arsenicals as compared with untreated cells. These results suggest that trivalent arsenicals inhibit insulin-stimulated glucose uptake by interfering with the PKB/Akt-dependent mobilization of GLUT4 transporters in adipocytes. This mechanism may be, in part, responsible for the development of type-2 diabetes in individuals chronically exposed to iAs.