Apigenin inhibits TGF-ß-induced VEGF expression in human prostate carcinoma cells via a Smad2/3- and Src-dependent mechanism.

Cancer progression relies on establishment of the blood supply necessary for tumor growth and ultimately metastasis. Prostate cancer mortality is primarily attributed to development of metastases rather than primary, organ-confined disease. Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis in prostate tissue. Our previous studies have demonstrated that the chemopreventive bioflavonoid apigenin inhibited hypoxia-induced elevation of VEGF production at low oxygen conditions characteristic for solid tumors. Low oxygen (hypoxia) and transforming growth factor-ß (TGF-ß) are two major factors responsible for increased VEGF secretion. In the present study, experiments were performed to investigate the inhibitory effect of apigenin on TGF-ß-induced VEGF production and the mechanisms underlying this action. Our results demonstrate that VEGF expression is induced by TGF-ß1 in human prostate cancer PC3-M and LNCaP C4-2B cells, and treatment with apigenin markedly decreased VEGF production. Additionally, apigenin inhibited TGF-ß1-induced phosphorylation and nuclear translocation of Smad2 and Smad3. Further experiments demonstrated that specific transient knockdown of Smad2 or Smad3 blunted apigenin's effect on VEGF expression. We also found that apigenin inhibited Src, FAK, and Akt phosphorylation in PC3-M and LNCaP C4-2B cells. Furthermore, constitutively active Src reversed the inhibitory effect of apigenin on VEGF expression and Smad2/3 phosphorylation. Taken together, our results suggest that apigenin inhibits prostate carcinogenesis by modulating TGF-ß-activated pathways linked to cancer progression and metastases, in particular the Smad2/3 and Src/FAK/Akt pathways. These findings provide new insights into molecular pathways targeted by apigenin, and reveal a novel molecular mechanism underlying the antiangiogenic potential of apigenin.

Apigenin, a chemopreventive bioflavonoid, induces AMP-activated protein kinase activation in human keratinocytes.

AMP-activated protein kinase (AMPK) is a cellular energy sensor that is conserved in eukaryotes. Although AMPK is traditionally thought to play a major role in the regulation of cellular lipid and protein metabolism, recent discoveries reveal that AMPK inhibits mammalian target of rapamycin (mTOR) signaling and connects with several tumor suppressors such as liver kinase B1 (LKB1), p53, and tuberous sclerosis complex 2 (TSC2), indicating that AMPK may be a potential target for cancer prevention and treatment. For the first time, we demonstrated that apigenin, a naturally occurring nonmutagenic flavonoid, induced AMPK activation in human keratinocytes (both cultured HaCaT cell line and primary normal human epidermal keratinocytes). Through experiments with over-expression of constitutively active Akt and knockdown of LKB1 expression by siRNAs, we further found that the activation of AMPK by apigenin was not dependent on its inhibition of Akt, and was independent of the activation of upstream kinase LKB1. Instead, another upstream kinase of AMPK, calcium/calmodulin-dependent protein kinase kinase-ß (CaMKKß), was required for apigenin-induced AMPK activation. We have demonstrated that knockdown of CaMKKß expression by siRNA or inhibition of CaMKKß activity by either CaMKK inhibitor STO-609 or BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester; a chelator of intracellular Ca(2+)) prevented apigenin-induced AMPK activation. Apigenin-induced AMPK activation inhibited mTOR signaling and further induced autophagy in human keratinocytes. These results suggest that one of the mechanisms by which apigenin exerts its chemopreventive action may be through activation of AMPK and induction of autophagy in human keratinocytes.

UVB radiation-induced ß-catenin signaling is enhanced by COX-2 expression in keratinocytes.

UVB radiation is the major carcinogen responsible for skin carcinogenesis, thus elucidation of the molecular pathways altered in skin in response to UVB would reveal novel targets for therapeutic intervention. It is well established that UVB leads to upregulation of cyclooxygenase 2 (COX-2) in the skin which contributes to skin carcinogenesis. Overexpression of COX-2 has been shown to promote colon cancer cell growth through ß-catenin signaling, however, little is known about the connection between UVB, COX-2, and ß-catenin in the skin. In the present study, we have identified a novel pathway in which UVB induces ß-catenin signaling in keratinocytes, which is modulated by COX-2 expression. Exposure of the mouse 308 keratinocyte cell line (308 cells) and primary normal human epidermal keratinocytes (NHEKs) to UVB resulted in increased protein levels of both N-terminally unphosphorylated and total ß-catenin. In addition, we found that UVB-enhanced ß-catenin-dependent TOPflash reporter activity and expression of a downstream ß-catenin target gene. We demonstrated that UVB-induced ß-catenin signaling is modulated by COX-2, as treatment of keratinocytes with the specific COX-2 inhibitor NS398 blocked UVB induction of ß-catenin. Additionally, ß-catenin target gene expression was reduced in UVB-treated COX-2 knockout (KO) MEFs compared to wild-type (WT) MEFs. Furthermore, epidermis from UVB-exposed SKH-1 mice exhibited increased N-terminally unphosphorylated and total ß-catenin protein levels and increased staining for total ß-catenin, and both responses were reduced in COX-2 heterozygous mice. Taken together, these results suggest a novel pathway in which UVB induces ß-catenin signaling in keratinocytes which is enhanced by COX-2 expression.

Apigenin down-regulates the hypoxia response genes: HIF-1α, GLUT-1, and VEGF in human pancreatic cancer cells.

BACKGROUND: The flavonoid apigenin exhibits anti-proliferative and anti-angiogenic activities. Our objective was to evaluate the effect of apigenin on hypoxia responsive genes important in pancreatic cancer cell proliferation. MATERIALS AND METHODS: Immunohistochemistry for GLUT-1 expression was conducted on human pancreatic cancer samples and adjacent controls. Real-time RT-PCR, Western blot analysis, and enzyme-linked immunosorbent assay (ELISA) were conducted on CD18 and S2-013 human pancreatic cancer cells treated with apigenin (0-50 µM) in normoxic and hypoxic conditions to evaluate HIF-1α, GLUT-1, and VEGF mRNA and protein expression and secretion. RESULTS: GLUT-1 expression was significantly increased in pancreatic adenocarcinoma samples versus adjacent controls (P < 0.001). Hypoxic conditions induced HIF-1α, GLUT-1, and VEGF protein expression in both CD18 and S2-013 pancreatic cancer cells. Apigenin (50 µM) blocked hypoxia induced up-regulation of all three proteins in both cell lines. Apigenin also impeded hypoxia-mediated induction of GLUT-1 and VEGF mRNA in both cell lines (P < 0.05). CONCLUSIONS: Apigenin inhibits HIF-1α, GLUT-1, and VEGF mRNA and protein expression in pancreatic cancer cells in both normoxic and hypoxic conditions. This may account for the mechanism of apigenin's anti-proliferative and anti-angiogenic effects and further supports the potential of apigenin as a future chemopreventive agent for pancreatic cancer.

Apigenin prevents UVB-induced cyclooxygenase 2 expression: coupled mRNA stabilization and translational inhibition.

Cyclooxygenase 2 (COX-2) is a key enzyme in the conversion of arachidonic acid to prostaglandins, and COX-2 overexpression plays an important role in carcinogenesis. Exposure to UVB strongly increased COX-2 protein expression in mouse 308 keratinocytes, and this induction was inhibited by apigenin, a nonmutagenic bioflavonoid that has been shown to prevent mouse skin carcinogenesis induced by both chemical carcinogens and UV exposure. Our previous study suggested that one pathway by which apigenin inhibits UV-induced and basal COX-2 expression is through modulation of USF transcriptional activity in the 5' upstream region of the COX-2 gene. Here, we found that apigenin treatment also increased COX-2 mRNA stability, and the inhibitory effect of apigenin on UVB-induced luciferase reporter gene activity was dependent on the AU-rich element of the COX-2 3'-untranslated region. Furthermore, we identified two RNA-binding proteins, HuR and the T-cell-restricted intracellular antigen 1-related protein (TIAR), which were associated with endogenous COX-2 mRNA in 308 keratinocytes, and apigenin treatment increased their localization to cell cytoplasm. More importantly, reduction of HuR levels by small interfering RNA inhibited apigenin-mediated stabilization of COX-2 mRNA. Cells expressing reduced TIAR showed marked resistance to apigenin's ability to inhibit UVB-induced COX-2 expression. Taken together, these results indicate that in addition to transcriptional regulation, another mechanism by which apigenin prevents COX-2 expression is through mediating TIAR suppression of translation.

Enhancement of p53 expression in keratinocytes by the bioflavonoid apigenin is associated with RNA-binding protein HuR.

We have reported previously that apigenin, a naturally occurring nonmutagenic flavonoid, increased wild-type p53 protein expression in the mouse keratinocyte 308 cell line by a mechanism involving p53 protein stabilization. Here we further demonstrated that the increase in p53 protein level induced by apigenin treatment of 308 keratinoyctes was not the result of enhanced transcription, mRNA stabilization or cytoplasmic export of p53 mRNA. Instead, biosynthetic labeling showed that apigenin increased nascent p53 protein synthesis by enhancing p53 translation. The AU-rich element (ARE) within the 3'-untranslated region (UTR) of p53 mRNA was found to be responsible for apigenin's ability to increase p53 translation, as demonstrated in studies wherein the 3'-UTR of p53 mRNA containing the ARE was fused downstream of a luciferase reporter gene. Furthermore, apigenin treatment increased the level of association of the RNA binding protein HuR with endogenous p53 mRNA. Apigenin treatment also augmented HuR translocation into the cytoplasm. Overexpression of HuR enhanced apigenin-induced p53 protein expression in 308 keratinocytes, whereas siRNA-mediated HuR reduction suppressed apigenin-induced p53 protein expression and de novo translation of p53. Moreover, apigenin treatment of cells induced p16 protein expression, which in turn was correlated with cytoplasmic localization of HuR induced by apigenin. Overall, these findings indicate that, in addition to modulating p53 protein stability, one of the mechanisms by which apigenin induces p53 protein expression is enhancement of translation through the RNA binding protein HuR.

Inhibition of HIF-1 alpha and VEGF expression by the chemopreventive bioflavonoid apigenin is accompanied by Akt inhibition in human prostate carcinoma PC3-M cells.

Progression of cancer leads to hypoxic solid tumors that mount specific cell signaling responses to low oxygen conditions. An important objective of anti-cancer therapy is the development of new drugs that suppress hypoxic responses in solid tumors. Apigenin is a natural flavone that has been shown to have chemopreventive and/or anti-cancer properties against a number of tumor types. However, the mechanisms underlying apigenin's chemopreventive properties are not yet completely understood. In this study, we have investigated the effects of apigenin on expression of hypoxia-inducible factor-1 (HIF-1) in human metastatic prostate PC3-M cancer cells. We found that hypoxia induced a time-dependent increase in the level of HIF-1alpha subunit protein in PC3-M cells, and treatment with apigenin markedly decreased HIF-1alpha expression under both normoxic and hypoxic conditions. Further, apigenin prevented the activation of the HIF-1 downstream target gene vascular endothelial growth factor (VEGF). We then showed that apigenin inhibited expression of HIF-1alpha by reducing stability of the protein as well as by reducing the level of HIF-1alpha mRNA. We also found that apigenin inhibited Akt and GSK-3beta phosphorylation in PC3-M cells. Further experiments demonstrated that constitutively active Akt blunted the effect of apigenin on HIF-1alpha expression. Taken together, our results identify apigenin as a bioflavonoid that inhibits hypoxia-activated pathways linked to cancer progression in human prostate cancer, in particular the PI3K/Akt/GSK-3 pathway. Further studies on the mechanism of action of apigenin will likely provide new insight into its applicability for pharmacologic targeting of HIF-1alpha for cancer therapeutic or chemopreventive purposes.

Geminin is overexpressed in human pancreatic cancer and downregulated by the bioflavanoid apigenin in pancreatic cancer cell lines.

Pancreatic adeniocarcinoma is among the deadliest of human cancers. Apigenin, an antitumor flavonoid, inhibits pancreatic cancer cell proliferation in vitro. Geminin is a recently identified novel protein that plays a critical role in preventing abnormal DNA replication by binding to and inhibiting the essential replication factor Cdt1. Microarray analysis identified geminin to be downregulated in pancreatic cancer cells treated with apigenin. Therefore, we investigated the effects of apigenin on geminin expression and other proteins involved in replication (Cdc6, Cdt1, and MCM7) in pancreatic cancer cell lines CD18 and S2013. Real time RT-PCR and western blotting analysis showed that geminin expression is downregulated by apigenin at both mRNA and protein levels. Furthermore, treatment of cells with proteosome inhibitor MG132 reversed the downregulation of geminin by apigenin, supporting our hypothesis that the degradation pathway is another mechanism by which apigenin affects geminin expression. Apigenin treatment also resulted in downregulation of Cdc6 at both mRNA and protein levels. However, Cdt1 and MCM7 expression was not affected in apigenin-treated cells. The effect of apigenin treatment on geminin promoter activity was measured by transient transfection of Hela cells with a reporter gene, demonstrating that apigenin inhibited geminin promoter activity. Geminin expression was also evaluated in human pancreatic tissue (n = 15) by immunohistochemistry and showed that geminin is overexpressed in human pancreatic cancer compared to normal adjacent pancreatic tissue. In conclusion, our studies demonstrated that geminin is overexpressed in human pancreatic cancer and downregulated by apigenin which may contribute to the antitumor effect of this natural flavonoid.

Inhibition of mTOR by apigenin in UVB-irradiated keratinocytes: A new implication of skin cancer prevention.

Ultraviolet B (UVB) radiation is the major environmental risk factor for developing skin cancer, the most common cancer worldwide, which is characterized by aberrant activation of Akt/mTOR (mammalian target of rapamycin). Importantly, the link between UV irradiation and mTOR signaling has not been fully established. Apigenin is a naturally occurring flavonoid that has been shown to inhibit UV-induced skin cancer. Previously, we have demonstrated that apigenin activates AMP-activated protein kinase (AMPK), which leads to suppression of basal mTOR activity in cultured keratinocytes. Here, we demonstrated that apigenin inhibited UVB-induced mTOR activation, cell proliferation and cell cycle progression in mouse skin and in mouse epidermal keratinocytes. Interestingly, UVB induced mTOR signaling via PI3K/Akt pathway, however, the inhibition of UVB-induced mTOR signaling by apigenin was not Akt-dependent. Instead, it was driven by AMPK activation. In addition, mTOR inhibition by apigenin in keratinocytes enhanced autophagy, which was responsible, at least in part, for the decreased proliferation in keratinocytes. In contrast, apigenin did not alter UVB-induced apoptosis. Taken together, our results indicate the important role of mTOR inhibition in UVB protection by apigenin, and provide a new target and strategy for better prevention of UV-induced skin cancer.

Chemopreventive apigenin controls UVB-induced cutaneous proliferation and angiogenesis through HuR and thrombospondin-1.

Plant flavonoid apigenin prevents and inhibits UVB-induced carcinogenesis in the skin and has strong anti-proliferative and anti-angiogenic properties. Here we identify mechanisms, by which apigenin controls these oncogenic events. We show that apigenin acts, at least in part, via endogenous angiogenesis inhibitor, thrombospondin-1 (TSP1). TSP1 expression by the epidermal keratinocytes is potently inhibited by UVB. It inhibits cutaneous angiogenesis and UVB-induced carcinogenesis. We show that apigenin restores TSP1 in epidermal keratinocytes subjected to UVB and normalizes proliferation and angiogenesis in UVB-exposed skin. Importantly, reconstituting TSP1 anti-angiogenic function in UVB-irradiated skin with a short bioactive peptide mimetic representing exclusively its anti-angiogenic domain reproduced the anti-proliferative and anti-angiogenic effects of apigenin. Cox-2 and HIF-1α are important mediators of angiogenesis. Both apigenin and TSP1 peptide mimetic attenuated their induction by UVB. Finally we identified the molecular mechanism, whereby apigenin did not affect TSP1 mRNA, but increased de novo protein synthesis. Knockdown studies implicated the RNA-binding protein HuR, which controls mRNA stability and translation. Apigenin increased HuR cytoplasmic localization and physical association with TSP1 mRNA causing de novo TSP1 synthesis. HuR cytoplasmic localization was, in turn, dependent on CHK2 kinase. Together, our data provide a new mechanism, by which apigenin controls UVB-induced carcinogenesis.

Targeting the PI3K/Akt/mTOR axis by apigenin for cancer prevention.

Natural products are important sources of anti-cancer lead molecules, and high dietary consumption of fruits and vegetables is associated with a reduced risk of certain cancers. Many efforts have been devoted to identifying and developing plant-derived dietary constituents as chemopreventive agents. Among them, apigenin, a naturally occurring flavonoid found in a variety of fruits and leafy vegetables, has been shown to possess remarkable anti-oxidant, anti-inflammatory and anti-carcinogenic properties. This review summarizes the anti-cancer and chemopreventive effects of apigenin at cellular and molecular levels, its chemical structure and properties, with focus on mechanism related to apigenin's inhibition of the PI3K/Akt/mTOR signaling pathways.

The desmosomal armadillo protein plakoglobin regulates prostate cancer cell adhesion and motility through vitronectin-dependent Src signaling.

Plakoglobin (PG) is an armadillo protein that associates with both classic and desmosomal cadherins, but is primarily concentrated in mature desmosomes in epithelia. While reduced levels of PG have been reported in localized and hormone refractory prostate tumors, the functional significance of these changes is unknown. Here we report that PG expression is reduced in samples of a prostate tumor tissue array and inversely correlated with advancing tumor potential in 7 PCa cell lines. Ectopically expressed PG enhanced intercellular adhesive strength, and attenuated the motility and invasion of aggressive cell lines, whereas silencing PG in less tumorigenic cells had the opposite effect. PG also regulated cell-substrate adhesion and motility through extracellular matrix (ECM)-dependent inhibition of Src kinase, suggesting that PG's effects were not due solely to increased intercellular adhesion. PG silencing resulted in elevated levels of the ECM protein vitronectin (VN), and exposing PG-expressing cells to VN induced Src activity. Furthermore, increased VN levels and Src activation correlated with diminished expression of PG in patient tissues. Thus, PG may inhibit Src by keeping VN low. Our results suggest that loss of intercellular adhesion due to reduced PG expression might be exacerbated by activation of Src through a PG-dependent mechanism. Furthermore, PG down-regulation during PCa progression could contribute to the known VN-dependent promotion of PCa invasion and metastasis, demonstrating a novel functional interaction between desmosomal cell-cell adhesion and cell-substrate adhesion signaling axes in prostate cancer.

The chemopreventive bioflavonoid apigenin inhibits prostate cancer cell motility through the focal adhesion kinase/Src signaling mechanism.

Prostate cancer mortality is primarily attributed to metastatic rather than primary, organ-confined disease. Acquiring a motile and invasive phenotype is an important step in development of tumors and ultimately metastasis. This step involves remodeling of the extracellular matrix and of cell-matrix interactions, cell movement mediated by the actin cytoskeleton, and activation of focal adhesion kinase (FAK)/Src signaling. Epidemiologic studies suggest that the metastatic behavior of prostate cancer may be an ideal target for chemoprevention. The natural flavone apigenin is known to have chemopreventive properties against many cancers, including prostate cancer. Here, we study the effect of apigenin on motility, invasion, and its mechanism of action in metastatic prostate carcinoma cells (PC3-M). We found that apigenin inhibits PC3-M cell motility in a scratch-wound assay. Live cell imaging studies show that apigenin diminishes the speed and affects directionality of cell motion. Alterations in the cytoskeleton are consistent with impaired cell movement in apigenin-treated cells. Apigenin treatment leads to formation of "exaggerated filopodia," which show accumulation of focal adhesion proteins at their tips. Furthermore, apigenin-treated cells adhere more strongly to the extracellular matrix. Additionally, apigenin decreases activation of FAK and Src, and phosphorylation of Src substrates FAK Y576/577 and Y925. Expression of constitutively active Src blunts the effect of apigenin on cell motility and cytoskeleton remodeling. These results show that apigenin inhibits motility and invasion of prostate carcinoma cells, disrupts actin cytoskeleton organization, and inhibits FAK/Src signaling. These studies provide mechanistic insight into developing novel strategies for inhibiting prostate cancer cell motility and invasiveness.

Dietary genistein inhibits metastasis of human prostate cancer in mice.

Dietary genistein has been linked to lower prostate cancer (PCa) mortality. Metastasis is the ultimate cause of death from PCa. Cell detachment and invasion represent early steps in the metastatic cascade. We had shown that genistein inhibits PCa cell detachment and cell invasion in vitro. Genistein-mediated inhibition of activation of focal adhesion kinase (FAK) and of the p38 mitogen-activated protein kinase (MAPK)-heat shock protein 27 (HSP27) pathway has been shown by us to regulate PCa cell detachment and invasion effects, respectively. To evaluate the antimetastatic potential of genistein, we developed an animal model suited to evaluating antimetastatic drug efficacy. Orthotopically implanted human PC3-M PCa cells formed lung micrometastasis by 4 weeks in >80% of inbred athymic mice. Feeding mice dietary genistein before implantation led to blood concentrations similar to those measured in genistein-consuming men. Genistein decreased metastases by 96%, induced nuclear morphometric changes in PC3-M cells indicative of increased adhesion (i.e., decreased detachment) but did not alter tumor growth. Genistein increased tumor levels of FAK, p38 MAPK, and HSP27 "promotility" proteins. However, the ratio of phosphorylated to total protein trended downward, indicating a failure to increase relative amounts of activated protein. This study describes a murine model of human PCa metastasis well suited for testing antimetastatic drugs. It shows for the first time that dietary concentrations of genistein can inhibit PCa cell metastasis. Increases in promotility proteins support the notion of cellular compensatory responses to antimotility effects induced by therapy. Studies of antimetastatic efficacy in man are warranted and are under way.

Enhancement of UVB-induced apoptosis by apigenin in human keratinocytes and organotypic keratinocyte cultures.

Topical application of the bioflavonoid 4',5,7-trihydroxyflavone (apigenin) to mouse skin effectively reduces the incidence and size of skin tumors caused by UVB exposure. The ability to act as a chemopreventive compound indicates that apigenin treatment alters the molecular events initiated by UVB exposure; however, the effects of apigenin treatment on UVB-irradiated keratinocytes are not fully understood. In the present study, we have used three models of human keratinocytes to study the effect of apigenin treatment on UVB-induced apoptosis: HaCaT human keratinocyte cells, primary keratinocyte cultures isolated from human neonatal foreskin, and human organotypic keratinocyte cultures. Each keratinocyte model was exposed to a moderate dose of UVB (300-1,000 J/m(2)), then treated with apigenin (0-50 micromol/L), and harvested to assess apoptosis by Western blot analysis for poly(ADP)ribose polymerase cleavage, annexin-V staining by flow cytometry, and/or the presence of sunburn cells. Apigenin treatment enhanced UVB-induced apoptosis >2-fold in each of the models tested. When keratinocytes were exposed to UVB, apigenin treatment stimulated changes in Bax localization and increased the release of cytochrome c from the mitochondria compared with UVB exposure alone. Overexpression of the antiapoptotic protein Bcl-2 and expression of a dominant-negative form of Fas-associated death domain led to a reduction in the ability of apigenin to enhance UVB-induced apoptosis. These results suggest that enhancement of UVB-induced apoptosis by apigenin treatment involves both the intrinsic and extrinsic apoptotic pathways. The ability of apigenin to enhance UVB-induced apoptosis may explain, in part, the photochemopreventive effects of apigenin.

Apigenin inhibits the GLUT-1 glucose transporter and the phosphoinositide 3-kinase/Akt pathway in human pancreatic cancer cells.

OBJECTIVES: The antiproliferative mechanisms of flavonoid drugs inpancreatic cancer cells remain unclear. In this study, we evaluated the effects of the flavonoid apigenin on glucose uptake, on the expression of the glucose transporter 1 (GLUT-1), and on the phosphoinositide 3-kinase (PI3K)/Akt pathway in human pancreatic cancer cells. METHODS: Human pancreatic cancer cells were treated with apigenin and then underwent glucose uptake assays. Real-time reverse transcription-polymerase chain reaction and Western blot analysis were conducted to evaluate GLUT-1 and pAkt expression in CD18 and S2-013 human pancreatic cancer cells after treatment with apigenin or PI3K inhibitors (LY294002 and wortmannin). RESULTS: Apigenin (0-100 microM) significantly inhibited, in a dose-dependent fashion, glucose uptake in CD18 and S2-013 human pancreatic cancer cell lines. Apigenin inhibited both GLUT-1 mRNA and protein expression in a concentration- and time-dependent fashion. The PI3K inhibitors, like apigenin, downregulated both GLUT-1 mRNA and protein expression. CONCLUSIONS: Our results demonstrate that the flavonoid apigenin decreases glucose uptake and downregulates the GLUT-1 glucose transporter in human pancreatic cancer cells. In addition, the inhibitory effects of apigenin and the PI3K inhibitors on GLUT-1 are similar, indicating that the PI3K/Akt pathway is involved in mediating apigenin's effects on downstream targets such as GLUT-1.

Modulation of UVB-induced and basal cyclooxygenase-2 (COX-2) expression by apigenin in mouse keratinocytes: role of USF transcription factors.

Apigenin is a bioflavonoid with chemopreventive activity against UV- or chemically-induced mouse skin tumors. To further explore the mechanism of apigenin's chemopreventive activity, we determined whether apigenin inhibited UVB-mediated induction of cyclooxygenase-2 (COX-2) expression in mouse and human keratinocytes. Apigenin suppressed the UVB-induced increase in COX-2 protein and mRNA in mouse and human keratinocyte cell lines. UVB radiation of keratinocytes transfected with a mouse COX-2 promoter/luciferase reporter plasmid resulted in a threefold increase in transcription from the promoter, and apigenin inhibited the UV-induced promoter activity at doses of 5-50 microM. Transient transfections with COX-2 promoter deletion constructs and COX-2 promoter constructs containing mutations in specific enhancer elements indicated that the effects of UVB required intact Ebox and ATF/CRE response elements. Electrophoretic mobility shift assays with supershifting antibodies were used to identify USF-1, USF-2, and CREB as proteins binding to the ATF/CRE-Ebox responsive element of the COX-2 promoter. Keratinocytes co-transfected with the COX-2 luciferase reporter and a USF-2 expression vector, alone or in combination with a USF-1 expression vector, exhibited enhanced promoter activity in both UVB-irradiated and nonirradiated cultures. However, COX-2 promoter activity was inhibited in keratinocytes co-transfected with USF-1 alone. Finally, we present data showing that the suppressive effect of apigenin on COX-2 expression could be reversed by co-expression of USF-1 and USF-2. These results suggest that one pathway by which apigenin inhibits COX-2 expression is through modulation of USF transcriptional activity.

Do truncated cyclins contribute to aberrant cyclin expression in cancer?

Cyclin overexpression is found in several types of cancer. Genetic events that place cyclin genes under the control of active promoters or that increase cyclin gene copy number account for most instances of cyclin overexpression. New paradigms for aberrant cyclin expression have been suggested by studies showing that truncated cyclins are expressed in specific subsets of cancer. The altered cyclins lack regulatory sequences (compared to the wild-type protein) that modulate their stability, subcellular localization or cdk-associated kinase activity. In this communication, we review the current literature and assess the role of truncated cyclins D, E, A, B, C and virus encoded-cyclin D (K-cyclin) in the development of cancer. We also report the molecular characteristics, expression patterns and if available, prognostic significance of these proteins.

Inhibition of TPA-induced cyclooxygenase-2 (COX-2) expression by apigenin through downregulation of Akt signal transduction in human keratinocytes.

Apigenin is a nonmutagenic bioflavonoid that has been shown to be an inhibitor of mouse skin carcinogenesis induced by the two-stage regimen of initiation and promotion with dimethylbenzanthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). These DMBA/TPA-induced squamous cell carcinomas overexpress cyclooxygenase-2 (COX-2). Cyclooxygenases are key enzymes required for prostaglandin (PG) synthesis, converting the arachidonic acid (AA) released by phospholipase A2 into prostaglandins. A large body of evidence indicates that the inducible form of cyclooxygenase, COX-2, is involved in tumor promotion and carcinogenesis in a wide variety of tissue types, including colon, breast, lung, and skin. In the present study, we have determined that apigenin inhibited the TPA-induced increase in COX-2 protein and mRNA in the human keratinocyte cell line; HaCaT. The induction of COX-2 elicited by TPA correlated with increased activation of Akt kinase and cell treatment with the PI3 kinase inhibitor, LY294002, blocked TPA induction of COX-2. In cells treated with TPA and apigenin, the inhibition of COX-2 expression correlated with inhibition of Akt kinase activation. Apigenin-mediated inhibition of TPA-induced COX-2 expression was reversed by transient transfection with constitutively active Akt (CA-Akt). Chemical inhibitors of MEK (PD98059), p38 (SB202190), but not JNK (SP600125) blocked TPA induction of COX-2 although apigenin did not inhibit TPA-mediated COX-2 expression through these pathways. The TPA-induced release of AA from HaCaT cells was also inhibited by cell treatment with apigenin. These data show that apigenin inhibits TPA-mediated COX-2 expression by blocking signal transduction of Akt and that apigenin also blocks AA release, which may contribute to its chemopreventive activity.

A p21(waf1)-independent pathway for inhibitory phosphorylation of cyclin-dependent kinase p34(cdc2) and concomitant G(2)/M arrest by the chemopreventive flavonoid apigenin.

Apigenin, a nonmutagenic flavonoid, has been shown to inhibit ultraviolet light-induced skin tumorigenesis when topically applied to mouse skin. Our previous studies have shown that apigenin treatment of cultured mouse keratinocytes induces G(2)/M arrest accompanied by an increase in p53 protein stability and expression of p21(waf1). In this study, we determined whether the G(2)/M arrest induced by apigenin was dependent upon the presence of the cyclin dependent kinase inhibitor p21(waf1). We exposed WWT.8 (p21(waf1) wild-type) and WKO.16 (p21(waf1) null) mouse keratinocytes to various doses of apigenin for 24 h and observed G(2)/M arrest in both cell lines, thereby establishing that the apigenin-induced G(2)/M arrest was p21(waf1) independent. A 4-h treatment with apigenin induced increases in p53 protein level by sixfold and tenfold in the WWT.8 p21(waf1) wild-type cells and WKO.16 p21(waf1) null cells, respectively. After 24 h in WWT.8 cells, p21(waf1) protein also was induced in a dose-dependent manner, but it was not expressed in WKO.16 keratinocytes. We then measured the effect of apigenin treatment on the mammalian homologue of the yeast cdc2 gene (p34(cdc2)) cyclin-dependent kinase and cyclin B1 (cycB1), because these proteins complex to regulate G(2)/M progression. Apigenin treatment decreased the protein level of p34(cdc2), and p34(cdc2) kinase activity was inhibited in both p21(waf1)(+/+) and p21(waf1)(-/-) cell lines by approximately 40%. The inhibition of p34(cdc2) kinase activity by apigenin treatment correlated with increasing levels of p34(cdc2) phosphorylation at Tyr15, a site in the p34(cdc2) kinase that undergoes inhibitory phosphorylation by Wee1 kinase. Apigenin treatment also had no effect on the protein level or activity of the competing phosphatase, cdc25c, which dephosphorylates p34(cdc2) kinase at Tyr15. Apigenin had little effect on the accumulation of cycB1 protein. These results supported the conclusion that G(2)/M arrest induced by apigenin was accompanied by inhibition of the p34(cdc2) cyclin-dependent kinase protein level and activity in a p21(waf1)-independent manner.