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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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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.

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-β (TGF-β) 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-β-induced VEGF production and the mechanisms underlying this action. Our results demonstrate that VEGF expression is induced by TGF-β1 in human prostate cancer PC3-M and LNCaP C4-2B cells, and treatment with apigenin markedly decreased VEGF production. Additionally, apigenin inhibited TGF-β1-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-β-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., (© 2013 Wiley Periodicals, Inc.)

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.

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 β-catenin signaling, however, little is known about the connection between UVB, COX-2, and β-catenin in the skin. In the present study, we have identified a novel pathway in which UVB induces β-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 β-catenin. In addition, we found that UVB-enhanced β-catenin-dependent TOPflash reporter activity and expression of a downstream β-catenin target gene. We demonstrated that UVB-induced β-catenin signaling is modulated by COX-2, as treatment of keratinocytes with the specific COX-2 inhibitor NS398 blocked UVB induction of β-catenin. Additionally, β-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 β-catenin protein levels and increased staining for total β-catenin, and both responses were reduced in COX-2 heterozygous mice. Taken together, these results suggest a novel pathway in which UVB induces β-catenin signaling in keratinocytes which is enhanced by COX-2 expression., (Copyright © 2011 Wiley Periodicals, Inc.)

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-β (CaMKKβ), was required for apigenin-induced AMPK activation. We have demonstrated that knockdown of CaMKKβ expression by siRNA or inhibition of CaMKKβ 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., (Copyright © 2011 Wiley Periodicals, Inc.)

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.

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., (Copyright © 2011. Published by Elsevier Inc.)

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.

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., (Copyright 2008 Wiley-Liss, Inc.)

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.

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.

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.

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.

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.

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.

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.

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.

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., ((c) 2005 Wiley-Liss, Inc.)

Apigenin is a nonmutagenic chemopreventive agent found in fruits and green vegetables. In this study, we used two different epithelial cell lines (308 mouse keratinocytes and HCT116 colon carcinoma cells) to determine the effect of apigenin on the mitogen-activated protein kinase (MAPK) cascade. Apigenin induced a dose-dependent phosphorylation of both extracellular signal-regulated protein kinase (ERK) and p38 kinase but had little effect on the phosphorylation of c-jun amino terminal kinase (JNK). We used immunoprecipitation-coupled kinase assays to show that apigenin increased the kinase activity of ERK and p38 but not JNK. Consistent with these results, we found that apigenin induced a 7.4-fold induction in the phosphorylation of Elk, the downstream phosphorylation target of ERK kinase. Similarly, apigenin induced a 3.2-fold induction in the phosphorylation of activating transcription factor-2, the downstream phosphorylation target of p38 kinase. Little change was observed in the phosphorylation of c-jun, the phosphorylation target of JNK. These data suggest that part of the chemopreventive activity of apigenin may be mediated by its ability to modulate the MAPK cascade.

Background: The proliferation of mural epithelial cells is a major cause of progressive cyst enlargement in autosomal-dominant polycystic kidney disease (ADPKD). Adenosine 3', 5' cyclic monophosphate (cAMP) stimulates the proliferation of cells from ADPKD cysts, but not cells from normal human kidney cortex (HKC), through the activation of protein kinase A (PKA), mitogen-activated protein kinase kinase (MEK), and extracellular signal-regulated kinase (ERK/MAPK). In the current study, we examined the signaling pathway between PKA and MEK in ADPKD and HKC cells., Methods: Primary cultures of human ADPKD and HKC cells were prepared from nephrectomy specimens. We determined the effects of cAMP and epidermal growth factor (EGF) on the activation of ERK, B-Raf and Raf-1 in ADPKD and HKC cells by immune kinase assay and Western blot., Results: 8-Br-cAMP increased phosphorylated ERK (2.7- +/- 0.6-fold, N = 7), and B-Raf kinase activity (3.6- +/- 1.1-fold, N = 5) in cells from ADPKD kidneys; levels of phosphorylated Raf-1 were not changed. Inhibition of PKA by H89 strikingly decreased cAMP-stimulated phosphorylation of ERK and B-Raf, and MAPK inhibition by PD98059 blocked the effect of the nucleotide to activate ERK. By contrast, in HKC cells 8-Br-cAMP did not activate B-Raf and ERK. EGF stimulated the phosphorylation of ERK and Raf-1 in both ADPKD and HKC cells, but had no effect on B-Raf. 8-Br-cAMP and EGF conjointly increased ERK activation above that of either agonist alone in ADPKD cells, and this combined effect was abolished by PD98059, indicating that ERK was activated by EGF- and cAMP-responsive cascades that converge at MAPK., Conclusion: cAMP activates ERK and increases proliferation of ADPKD epithelial cells, but not cells from normal human kidney cortex, through the sequential phosphorylation of PKA, B-Raf and MAPK in a pathway separate from, but complementary to, the classical receptor tyrosine kinase cascade. Consequently, cAMP and EGF have great potential to accelerate the progressive enlargement of renal cysts.

Exposure of mammalian cells to genotoxic stress results in activation of the c-jun amino-terminal kinase (JNK)-stress-activated protein kinase (SAPK) pathway and induction of DNA repair enzymes and cell cycle-regulatory proteins such as p53 and p21waf1. The p53 tumor suppressor protein transmits signals that activate p21waf1 gene expression. The p21waf1 protein then restricts cell-cycle progression, thereby allowing time for DNA repair to occur. In this study, we investigated the effects of modulation of the level of wild-type and mutant p53 protein on basal JNK1 activity in the A1-5 rat fibroblast cell line. This cell line contains a p53 gene coding for a temperature-sensitive p53 protein, which allows us to regulate the relative level of wild-type and mutant p53 protein produced in cells. Using the immune complex kinase assay to measure JNK1 activity, we demonstrated that cells expressing the wild-type-conformation p53 protein (when grown at 32.5 degrees C) exhibited a very low level of JNK1 activity. When cells were grown at 37 degrees C or 39 degrees C to express predominantly mutant p53 protein, basal level of JNK1 activity was significantly higher than at 32.5 degrees C. We also demonstrated protein-protein interactions between the p53, p21waf1, and JNK1 proteins in this cell line. Both wild-type p53 protein (expressed at 32.5 degrees C) and mutant p53(val135) protein (expressed at 37 degrees C and 39 degrees C) were present in immunocomplexes of JNK1 protein. Under conditions where wild-type p53 protein was present to induce p21waf1 expression (at 32.5 degrees C), a higher level of p21waf1 protein was also detected in the JNK1 immunocomplexes than in those at 37 degrees C and 39 degrees C. We next investigated the effect that co-association of p53 protein and p21waf1 protein would have on JNK1 activity. We measured basal levels of JNK1 activity in cells expressing wild-type p53 and p21waf1, or in p21waf1-null cells, and demonstrated that cells expressing both p53 and p21waf1 proteins exhibited an approximately threefold lower basal level of JNK1 activity when compared with p21waf1-null cells. To confirm that p21waf1 protein expression in cells resulted in reduced JNK1 activity, we transfected p21waf1-/- cells with a p21waf1 expression vector. We observed that JNK1 activity was inhibited after exogenous p21waf1 protein was expressed in these cells. Our results provide evidence for modulation of the JNK1 pathway by p53 and p21waf1 proteins and support the hypothesis that modulation of JNK1 activity occurred through protein-protein interactions between JNK1, p53, and p21waf1 proteins., (Copyright 2002 Wiley-Liss, Inc.)

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., (Copyright 2002 Wiley-Liss, Inc.)

The tumor suppressor protein p53 plays an important role in recognition of DNA damage and induction of subsequent cell cycle arrest. One of its target genes encodes the protein p21(WAF1), which is involved in mediation of growth arrest after DNA damage has occurred. Dibenzo[a,l]pyrene (DB[a,l]P) is a polycyclic aromatic hydrocarbon which is an exceptionally potent carcinogen. A reactive secondary metabolite of DB[a,l]P, the fjord region (-)-anti-11R,12S-dihydrodiol 13R,14S-epoxide [(-)-anti-DB[a,l]PDE] was used to investigate DNA damage via adduct formation and cell cycle arrest in human diploid fibroblast cell cultures (HDF). Synchronous HDF were exposed to increasing concentrations (0.014, 0.028 and 0.07 microM) of (-)-anti-DB[a,l]PDE and at 1, 12, 24 and 42 h after treatment cell pellets were analyzed for DNA adduct formation and cell cycle arrest. Exposure of HDF to 0.07 microM (-)-anti-DB[a,l]PDE caused a total DNA binding level of 113 pmol adducts/mg DNA (42 h after treatment). G(1) arrest was induced by this treatment, with 91% of the cells remaining in G(1) phase compared with the solvent-treated control cultures (50%) as analyzed by propidium iodide staining and flow cytometry. Further investigation of the percentage of cells in S phase by 5-bromo-2'-deoxyuridine incorporation confirmed the G(1) arrest in HDF treated with 0.07 microM (-)-anti-DB[a,l]PDE, with only 1.5% of the cells moving into S phase compared with 39% in the control 42 h after treatment. Induction of p53 and p21(WAF1) was demonstrated by western blot analysis.

The cellular response to cisplatin involves activation of multiple signal transduction pathways, including the mitogen-activated protein (MAP) kinase pathways. In this study, we compared the cisplatin-induced activation of two MAP kinases, c-jun N-terminal kinase 1 (JNK1) and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), in the cisplatin-sensitive ovarian carcinoma cell line A2780 and its derivative cisplatin-resistant cell lines CP70 and C200. Dose-dependent and time-dependent activation of JNK1 and ERK1/2 occurred in each of the three cell lines in response to cisplatin treatment. The requirement of higher concentrations of cisplatin for induction of maximum activation of JNK1 and ERK1/2 was correlated with increased levels of cisplatin resistance. In addition, inhibition of cisplatin-induced ERK activation, using the MAP/ERK kinase 1 synthetic inhibitor PD98059, resulted in enhanced sensitivity to cisplatin in all three cell lines. These results suggest that cisplatin-induced ERK1/2 activity is not responsible for the acquired cisplatin resistance in CP70 and C200 cells but rather provides a general cytoprotective effect in both cisplatin-sensitive and cisplatin-resistant cell lines. In conclusion, different patterns of cisplatin-induced JNK1 and ERK1/2 activation are observed in cell lines with different levels of cisplatin sensitivity, and inhibition of cisplatin-induced ERK1/2 activation enhances sensitivity to cisplatin in both cisplatin-sensitive and cisplatin-resistant cell lines.

The p53 tumor suppressor protein is a transcription factor that plays a major role in the DNA damage response. After DNA damage, p53 levels increase due primarily to stabilization of the protein. The molecular mechanisms leading to stabilization of p53 after DNA damage have not been completely elucidated. Recently we reported that cisplatin treatment activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) and that inhibition of ERK1/2 resulted in enhanced sensitivity to cisplatin. In the present study, we examined the potential role of ERK1/2 activation in regulation of the p53 response to cisplatin. In the ovarian carcinoma cell line A2780, inhibition of ERK1/2 activation with the mitogen-activated protein kinase/ERK kinase 1 (MEK1) inhibitor PD98059 resulted in decreased p53 protein half-life and diminished accumulation of p53 protein during exposure to cisplatin. We also demonstrated that p53 protein co-immunoprecipitated with ERK1/2 protein and was phosphorylated by activated recombinant murine ERK2 in vitro. Furthermore, PD98059 decreased the phosphorylation of p53 at serine 15 during cisplatin exposure, suggesting that ERK1/2 mediates in part phosphorylation of p53 during the cisplatin DNA response. These results strongly suggest that cisplatin-induced ERK activation is an up-stream regulator of the p53 response to DNA damage caused by cisplatin.

Apigenin, a common dietary flavonoid, has been shown to induce cell cycle arrest in both epidermal and fibroblast cells and inhibit skin tumorigenesis in murine models. The present study assessed the influence of apigenin on cell growth and the cell cycle in the human colon carcinoma cell lines SW480, HT-29, and Caco-2. Treatment of each cell line with apigenin (0-80 microM) resulted in a dose-dependent reduction in both cell number and cellular protein content, compared with untreated control cultures. DNA flow cytometric analysis indicated that treatment with apigenin resulted in G2/M arrest in all three cell lines in a time- and dose-dependent manner. Apigenin treatment (80 microM) for 48 h produced maximum G2/M arrest of 64%, 42%, and 26% in SW480 cells, HT-29 cells, and Caco-2 cells, respectively, in comparison with control cells (15%). The proportion of S-phase cells was not altered by apigenin treatment in each of the three cell lines. The G2/M arrest was reversible after 48 h of apigenin treatment in the most sensitive cell line SW480. The degree of G2/M arrest by apigenin was inversely correlated with the corresponding inhibition of cell growth measurements in all three cell lines (r = -0.626 to -0.917, P

Apigenin, a naturally occurring, non-mutagenic flavonoid, has been shown to inhibit UV-induced skin tumorigenesis in mice when topically applied. In this report we have used the mouse keratinocyte 308 cell line, which contains a wild-type p53 gene, to study the effect of apigenin treatment on p53 protein levels and the expression of its downstream partner, p21/waf1. Cells were treated with 70 microM apigenin for various times and levels of p53 and p21/waf1 protein were assessed by western blot analysis. The level of p53 protein was induced 27-fold after 4 h of apigenin treatment and levels remained elevated through 10 h of exposure. After 24 h of exposure to 70 microM apigenin, p53 protein levels returned to control levels. p21/waf1 protein levels increased approximately 1. 5-2-fold after 4 h and remained elevated at 24 h. To investigate the mechanism of p53 protein accumulation, we compared the half-life of p53 protein in vehicle- and apigenin-treated cells. Cells were incubated for 4 h in the presence of apigenin, then cycloheximide was added to inhibit further protein synthesis and p53 protein levels were measured by western blot. The half-life of p53 protein was found to be increased an average of 8-fold in apigenin-treated cells compared with vehicle-treated cells (t(1/2) = 131 min versus 16 min in apigenin- versus vehicle-treated cells, respectively). The mechanism of p53 protein stabilization is currently being investigated. To determine whether p53 was transcriptionally active, we also performed gel mobility shift assays and transient transfection studies using a luciferase plasmid under the control of the p21/waf1 promoter. Both p53 DNA-binding activity and transcriptional activation peaked after 24 h of exposure to apigenin. These studies suggest that apigenin may exert anti-tumorigenic activity by stimulating the p53-p21/waf1 response pathway.

Background: : Cellular proliferation is a key factor in the enlargement of renal cysts in autosomal dominant polycystic kidney disease (ADPKD). We determined the extent to which adenosine 3':5'-cyclic monophosphate (cAMP) may regulate the in vitro proliferation of cyst epithelial cells derived from human ADPKD cysts., Methods: : Epithelial cells from cysts of individuals with ADPKD and from normal human kidney cortex (HKC) of individuals without ADPKD were cultured. The effects of agonists and inhibitors on the rate of cellular proliferation and the activation of extracellular signal-regulated kinase (ERK1/2) were determined., Results: : 8-Br-cAMP (100 micromol/L) stimulated the proliferation of cells from eight different ADPKD subjects to 99.0% above baseline; proliferation was inhibited by protein kinase A (PKA) antagonists H-89 (97%) and Rp-cAMP (90%). Forskolin (10 micromol/L), which activates adenylyl cyclase, increased proliferation 124%, and receptor-mediated agonists arginine vasopressin, desmopressin, secretin, vasoactive intestinal polypeptide, and prostaglandin E2 stimulated proliferation 54.2, 56.3, 46.7, 37.1, and 48.3%, respectively. The mitogen extracellular kinase (MEK) inhibitor PD98059 completely inhibited ADPKD cell proliferation in response to cAMP agonists, but genistein, a receptor tyrosine kinase inhibitor, did not block cAMP-dependent proliferation. cAMP agonists increased the activity of ERK above control levels within five minutes. In contrast to ADPKD, proliferation and ERK activity of cells derived from normal HKC were not stimulated by cAMP agonists, although electrogenic Cl- secretion was increased by these agonists in both ADPKD and HKC cell monolayers., Conclusions: : We conclude that cAMP agonists stimulate the proliferation of ADPKD but not HKC epithelial cells through PKA activation of the ERK pathway at a locus distal to receptor tyrosine kinase. We suggest that the adenylyl cyclase signaling pathway may have a unique role in determining the rate of cyst enlargement in ADPKD through its actions to stimulate cellular proliferation and transepithelial solute and fluid secretion.

Exposure of mammalian cells to ultraviolet (UV) light and other DNA-damaging agents triggers the UV response which is characterized by induction of a large number of genes including c-fos, c-jun, and the genes for DNA repair enzymes and cell-cycle regulatory proteins such as p21 WAF1 and p53. Upon DNA damage, the p53 tumor suppressor protein transmits signals to restrict cell-cycle progression, thereby allowing time for DNA repair to occur. Cells also respond to genotoxic stress by activation of the jun N-terminal kinase (JNK)/stress-activated protein kinase pathway. In this report we investigated the effects of modulation of the level of wild-type and mutant p53 protein on basal and UV-inducible JNK activity. We used the A1-5 rat fibroblast cell line, which contains a p53 gene coding for a temperature-sensitive p53 protein, which allows us to regulate the relative level of wild-type and mutant p53 protein produced in a cell. We measured the relative levels of JNK activity in sham-irradiated and UV-irradiated cells by using the immune complex kinase assay and then computed the fold induction of JNK after UV exposure. We demonstrated that cells expressing p53 protein in the wild-type conformation (when grown at 32 degrees C) exhibited a very low level of JNK activity that was induced 14- to 16-fold by UVC irradiation. When cells were grown at 37 degrees C or 39 degrees C to express predominantly mutant p53 protein, basal JNK activity was significantly higher than at 32 degrees C. UVC irradiation of cells expressing mutant p53 protein resulted in JNK activation, although the overall fold-induction was only two-fold because JNK1 activity was already high in the sham-treated controls. UVB irradiation also induced JNK1 activity, although we again observed a relatively high level of basal JNK activity in sham-irradiated cells expressing mutant p53 protein compared with cells expressing wild-type p53. Control experiments confirmed that JNK1 basal activity was not affected by temperature alone. Western blot analysis of cell extracts indicated that expression of p21 WAF protein was significantly higher in cells expressing wild-type p53 protein and was associated with low basal levels of JNK1 activity. In contrast, cells expressing mutant p53 protein and very low levels of p21 WAF1 protein were found to have a higher level of basal JNK1 activity. We also observed a reduced ability to induce JNK1 after UV irradiation of several other cell lines with p53-mutant or p53-null genotypes. Our results provide evidence for a novel connection between p53 status and the basal level of JNK1, a critical enzyme in the stress-activated protein kinase family. In addition, these studies suggest that the presence of mutant p53 protein in a cell not only affects basal activity of JNK1 but also affects the ability of a cell to respond to UV-induced stress by transmitting signals via induction or activation of the JNK1 cascade.

Tumor necrosis factor-alpha (TNF alpha) can function as both an autocrine and a paracrine growth factor and may therefore play a role in ovarian tumor progression. TNF alpha initiates multiple cellular responses, many of which are mediated through the mitogen-activated protein kinase pathways, which transduce signals from the TNF alpha receptors through the cytoplasm to the nucleus, resulting in regulation of gene expression. We examined the role of c-jun N-terminal kinase 1 (JNK1) and extracellular signal-regulated protein kinase (ERK) 1 and 2 in the cellular growth response to TNF alpha in the ovarian carcinoma cell line UCI 101. JNK1 activity was increased to a maximum level ninefold above the basal level after 10-20 min of treatment with 10 ng/mL TNF alpha. A maximum threefold induction of ERK1/2 activity was observed after 1 min of treatment. At concentrations up to 100 ng/mL, TNF alpha had neither a stimulatory nor an inhibitory effect on growth of UCI 101 cells. However, inhibition of TNF alpha-induced ERK1/2 activity by the MAP/ERK kinase 1 inhibitor PD 98059 resulted in 60% inhibition of cell growth in TNF alpha-treated UCI 101 cells. This decrease in cell growth was accompanied by apoptosis, as demonstrated by the presence of a 180-bp DNA ladder. Thus, the inhibition of TNF alpha-induced ERK1/2 activity was associated with induction of apoptosis in the TNF alpha-resistant cell line UCI 101. Inhibition of TNF alpha-induced ERK1/2 activity was accompanied by a subsequent transient increase in TNF alpha-induced JNK1 activity. The significance of this increase with respect to apoptosis induction remains to be determined. These findings demonstrated that ERK1/2 activity can modulate cellular sensitivity to TNF alpha and suggested that the balance between the levels of ERK1/2 and JNK1 activation may be critical in the cellular growth response to TNF alpha.

Cisplatin treatment activates multiple signal transduction pathways, which can lead to several cellular responses including cell cycle arrest, DNA repair, survival, or apoptosis. We investigated the response of the mitogen-activated protein kinases, extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun-N-terminal kinase 1 (JNK1), and p38, to cisplatin treatment in the ovarian carcinoma cell line SK-OV-3. Cisplatin caused a late and prolonged induction in a dose-dependent manner of both ERK1/2 and JNK1 activity. ERK1/2 and JNK1 activities continued to increase in magnitude up to 24 h following initiation of cisplatin treatment. In contrast, cisplatin treatment had no effect on p38 activity. Transplatin failed to induce either ERK1/2 or JNK1 at 24 h, which suggests that the activation of these kinases was dependent on cisplatin-specific DNA damage. Treatment with cycloheximide resulted in inhibition of cisplatin-induced ERK1/2 activation, demonstrating that ERK1/2 activity induced by cisplatin was dependent on de novo protein synthesis. Furthermore, inhibition of cisplatin-induced ERK1/2 activity by PD 98059 caused enhanced cisplatin cytotoxicity. Similar enhanced cytotoxic effects of cisplatin were also observed following treatment with PD 98059 in the ovarian carcinoma cell line UCI 101. These observations indicate that ERK1/2 activation induced by cisplatin partially protects cells from cisplatin cytotoxicity. Continued investigation into the mechanism by which the ERK pathway and other signal transduction pathways modulate the response to cisplatin may be helpful in the development of new strategies for improving the therapeutic use of platinum drugs.

Exposure to solar ultraviolet (UV) radiation is believed to cause most human skin carcinomas. Despite the large body of evidence connecting UV exposure with skin cancer, the frequency and level of human exposure to repetitive doses of UV light will most likely continue for occupational and recreational reasons. By investigating the cellular response of keratinocytes to multiple, physiologically relevant doses of UV, we hope to better understand the processes involved in UV-induced skin cancer. In this study, we used a UV exposure model to investigate the cell-cycle response of keratinocytes exposed to multiple doses of UV-B/A radiation in which the UV-C component (wavelengths below 290 nm) had been filtered out. Our results indicated that exposure of asynchronous mouse keratinocytes to three doses of 200 J/m2 UV-B/A radiation at 30 min intervals produced an inhibition of DNA synthesis and S-phase arrest between 7 and 25 h after the last irradiation. The S-phase arrest was not due to a reduction in the level of cyclin E and A proteins but was accompanied by inhibition of cyclin-dependent kinase 2 (cdk2) activity. We observed a similar pattern of cdk2 inhibition induced by multiple UV-B/A irradiations in mouse embryo fibroblasts from p21WAF null mice, indicating that the inhibition of cdk2 was independent of p21WAF in these cells.

The biological effects of antioxidants are often considered in terms of their effects on oxygen or lipid radicals. However, antioxidants can also exert their effects through altering the cellular redox potential. Herein, we report that sulfur-containing antioxidants such as N-acetylcysteine and dimercaptopropanol induced apoptosis in several transformed cell lines and transformed primary cultures but not in normal cells. In contrast, chain-breaking antioxidants such as vitamin E lacked this activity. An increased glutathione level was not required for apoptosis; however, all apoptosis-inducing antioxidants elevated the total cellular thiol levels. Antioxidant-induced apoptosis required the p53 tumor suppressor gene. N-Acetylcysteine elevated p53 expression posttranscriptionally by increasing the rate of p53 mRNA translation rather than by altering the protein stability. The p53 induction occurred in normal cells. These observations indicate a redox sensor for p53 induction in vivo, with additional transformation-specific information being required for apoptosis. Manipulating p53-dependent apoptosis with nontoxic antioxidants may have a direct clinical application.

Jun N-terminal kinase (JNK1) is a member of a family of stress-activated protein kinases which are activated by many forms of stress including UV radiation, resulting in the phosphorylation of c-Jun, ATF-2, Elk-1 and p53. As UV-B radiation is mainly responsible for ultraviolet (UV)-induced skin cancers, we chose to elucidate JNK1 activation in keratinocytes which represent a UV-relevant cell system. We have demonstrated rapid activation of JNK1 in a keratinocyte cell line, C50, in response to multiple doses of UV-B irradiation. JNK1 activation occurred within 1 min, peaked by 10 min and returned to near basal levels within 2 h following the UV-B treatments. Our data provide the first evidence to show that keratinocytes do respond to multiple doses of the physiologically relevant UV-B radiation through rapid activation of the JNK1 pathway.

Apigenin is a plant flavonoid that has been shown to significantly inhibit ultraviolet-induced mouse skin tumorigenesis when applied topically and may be an alternative sunscreen agent for humans. A long-term goal of our laboratory is to elucidate the molecular mechanism or mechanism by which apigenin inhibits skin tumorigenesis. In a previous publication, we characterized the mechanism by which apigenin induced G2/M arrest in keratinocytes. More recent studies in our laboratory have provided evidence that apigenin can induce G1 arrest in addition to arresting cells at G2/M. Here we describe the mechanism of the apigenin-induced G1 arrest in human diploid fibroblasts (HDF). Treatment of asynchronous HDF for 24 h with 10-50 microM apigenin resulted in dose-dependent cell-cycle arrest at both the G0/G1 and G2/M phases as measured by flow cytometry. The G0/G1 arrest was more clearly defined by using HDF that were synchronized in G0 and then released from quiescence by replating at subconfluent densities in medium containing 10-70 microM apigenin. The cells were analyzed for cell-cycle progression or cyclin D1 expression 24 h later. A dose of apigenin as low as 10 microM reduced the percentage of cells in S phase by 20% compared with control cultures treated with solvent alone. Western blot analysis of apigenin-treated HDF indicated that cyclin D1 was expressed at higher levels than in untreated cells, which signifies that they were arrested in G1 phase rather than in a G0 quiescent state. The G1 arrest was further studied by cyclin-dependent kinase 2 (cdk2) immune complex-kinase assays of apigenin-treated asynchronous HDF, which demonstrated a dose-dependent inhibition of cdk2 by apigenin. Inhibition of cdk2 kinase activity in apigenin-treated cells was associated with the accumulation of the hypophosphorylated form of the retinoblastoma (Rb) protein as measured by western blot analysis. The cdk inhibitor p21/WAF1 was also induced in a dose-dependent manner, with a 22-fold induction of p21/WAF1 in 70 microM apigenin-treated cells. In conclusion, apigenin treatment produced a G1 cell-cycle arrest by inhibiting cdk2 kinase activity and the phosphorylation of Rb and inducing the cdk inhibitor p21/WAF1, all of which may mediate its chemopreventive activities in vivo. To our knowledge this is the first report of a chemopreventive agent inducing p21/WAF1, a known downstream effector of the p53 tumor suppressor protein.

Apigenin is a plant flavonoid which has been shown to significantly inhibit UV-induced mouse skin tumorigenesis when applied topically, and may represent an alternative sunscreen agent in humans. We have investigated the molecular mechanism(s) by which apigenin inhibits skin tumorigenesis. Initial studies examined the effects of apigenin on the cell cycle. DNA flow cytometric analysis indicated that culturing cells for 24 h in medium containing apigenin induced a G2/M arrest in two mouse skin derived cell lines, C50 and 308, as well as in human HL-60 cells. The G2/M arrest was fully reversible after an additional 24 h in medium without apigenin. We investigated the effects of apigenin on cyclin B1 and p34cdc2, since cyclin B1/p34cdc2 complexes regulate G2/M progression. Western blot and immune complex kinase assays using whole cell lysates from 308 and C50 cells treated for 24 h with 0-70 microM doses of apigenin demonstrated that apigenin treatment did not change the steady-state level of p34cdc2 protein, but did inhibit p34cdc2 H1 kinase activity in 308 cells. Western blot analysis showed that apigenin treatment of C50 cells and 308 cells inhibited the accumulation of cyclin B1 protein in a dose-dependent manner. The apigenin levels detected in cultured keratinocytes were relevant to those detected in epidermal cells of Sencar mice treated with tumor inhibitory doses of apigenin. In conclusion, we present evidence that apigenin induces a reversible G2/M arrest in cultured keratinocytes, the mechanism of which is in part due to inhibition of the mitotic kinase activity of p34cd2, and perturbation of cyclin B1 levels.

Considerable evidence suggests that dietary differences between populations account for a significant proportion of the variation in cancer occurrence in different parts of the world. A major problem has been identifying the particular dietary components which predispose or protect individuals against cancer. For example, the high rates of breast and colon cancer in the United States have been associated with numerous dietary patterns including high fat, high dietary energy, and low fruit and vegetable intakes. Our laboratories have attempted to identify mechanisms whereby diet may modify cancer and it is anticipated that future studies will determine which of these potential mechanisms may be relevant in humans. A promising lead in understanding the mechanism of high dietary fat/high dietary energy promotion of cancer was the impact of these diets on cellular protein kinase C (PKC). PKC is important in cellular signaling events which are critical to tumor promotion. Our studies demonstrated increased PKC activity and/or protein expression observed in epidermis and pancreatic epithelial cells of rodents fed high fat/energy diets. The inverse association between cancer at a number of sites and fruit and vegetable intake may be due to both micronutrient and non-nutrient components of fruits and vegetables. We have studied the prevention of skin tumor promotion by apigenin, a plant flavonoid. Apigenin may block several points in the process of tumor promotion, including inhibiting kinases, reducing transcription factors and regulating cell cycle. The complexity of our diets and the multitude of potential dietary effects which may be important in cancer development make this a fertile area for future study.

Exposure to solar ultraviolet (UV) light is a major cause of skin cancer, the most common human neoplasm. The earth's upper atmosphere absorbs the high energy UV-C wavelengths (100-280 nm), while allowing transmission of UV-B (280-320 nm) and UV-A (320-400 nm). It is therefore UV-B and to some extent UV-A, that contributes to most human skin malignancies. We report that the exposure of cultured keratinocytes or skin to UV-C radiation causes activation of MAP kinases (ERK and JNK). In contrast, the solar radiation associated with skin cancer (UV-B) was an ineffective activator of the ERK and JNK signal transduction pathways. Therefore, while exposure of epidermal cells to UV-C radiation under laboratory conditions causes marked activation of MAP kinase signal transduction pathways, only a low level of MAP kinase signaling is involved in the response of skin to biologically relevant solar radiation.

Mouse skin tumors contain activated c-H-ras oncogenes, often caused by point mutations at codons 12 and 13 in exon 1 and codons 59 and 61 in exon 2. Mutagenesis by the noncoding apurinic sites can produce G-->T and A-->T transversions by DNA misreplication with more frequent insertion of deoxyadenosine opposite the apurinic site. Papillomas were induced in mouse skin by several aromatic hydrocarbons, and mutations in the c-H-ras gene were determined to elucidate the relationship among DNA adducts, apurinic sites, and ras oncogene mutations. Dibenzo[a,l]pyrene (DB[a,l]P), DB[a,l]P-11,12-dihydrodiol, anti-DB[a,l]P-11,12-diol-13,14-epoxide, DB[a,l]P-8,9-dihydrodiol, 7,12-dimethylbenz[a]anthracene (DMBA), and 1,2,3,4-tetrahydro-DMBA consistently induced a CAA-->CTA mutation in codon 61 of the c-H-ras oncogene. Benzo[a]pyrene induced a GGC-->GTC mutation in codon 13 in 54% of tumors and a CAA-->CTA mutation in codon 61 in 15%. The pattern of mutations induced by each hydrocarbon correlated with its profile of DNA adducts. For example, both DB[a,l]P and DMBA primarily form DNA adducts at the N-3 and/or N-7 of deoxyadenosine that are lost from the DNA by depurination, generating apurinic sites. Thus, these results support the hypothesis that misreplication of unrepaired apurinic sites generated by loss of hydrocarbon-DNA adducts is responsible for transforming mutations leading to papillomas in mouse skin.

The tumor suppressor gene p53 is involved in controlling cell cycle checkpoint or triggering apoptosis. p53 may accomplish these roles by acting as a sequence-specific transcription factor. One of the downstream targets of p53 transcription control is the WAF1/CIP1 gene, whose gene product p21 interacts with several cyclins and cyclin-dependent kinases, resulting in inhibition of these kinases. In our previous studies, we have shown that the p53 protein level in mouse keratinocytes was elevated following UV-B/A irradiation. In this paper we further investigated the consequences of increased p53 protein level by characterizing p53 DNA-binding level and WAF1/CIP1 gene expression in UV-B/A-irradiated mouse keratinocytes. Consistent with the increased level of p53 protein, both p53 DNA-binding level and steady-state level of WAF1/CIP1 mRNA were elevated. We have demonstrated that the induction of WAF1/CIP1 mRNA was mediated by p53, since no WAF1/CIP1 induction was observed in p53-deficient cells upon UV-B exposure. These observations suggest an important role for the tumor suppressor gene p53 in the response of keratinocytes to the biologically relevant UV-B/A irradiation and in suppressing UV-induced skin cancer.

Several lines of evidence that indicate that mutation of the Ha-ras oncogene is the initiating event in mouse skin carcinogenesis. Keratinocytes known to possess a mutated Ha-ras have been shown to be resistant to differentiation. Thus, overstimulation of the Ha-ras signaling pathway appears to block normal keratinocyte differentiation, and we hypothesized that for normal keratinocytes to terminally differentiate, the Ha-ras signaling cascade must be turned off. In the present studies, we measured the level and activity state of Ha-ras p21 protein in cultured keratinocytes undergoing calcium-induced differentiation. We have employed Western blot analysis to demonstrate that Ha-ras p21 protein levels remain constant during primary newborn and adult keratinocyte differentiation. The overall level of Ha-ras p21 was higher in immortalized, benign, and malignant mouse keratinocyte cell lines than in normal keratinocytes but did not change within each cell type when subjected to differentiating conditions. The percentage of Ha-ras p21 protein in its active, GTP-bound form also remained unchanged during primary adult keratinocyte differentiation and in immortalized, benign, and malignant keratinocytes subjected to differentiating conditions. Our results indicate that terminal differentiation of primary adult mouse keratinocytes occurred in the presence of constant levels of Ha-ras p21-GTP, suggesting that the Ha-ras signaling pathway may be blocked at a point distal to a step involving the Ha-ras p21 protein itself.

The purpose of our research is to understand the biochemical basis for dietary enhancement of phorbol ester induced tumor promotion in mice fed high-fat (HF) diet, and the inhibition of promotion in mice fed diets restricted in energy from fat and carbohydrate (ER). The present study assessed the presence of protein kinase C (PKC) isoenzymes in the Sencar mouse epidermis by Western blot and determined the influence of diet on the isoenzymes found. Mice were fed control, HF (24.5% corn oil) or ER (60% of control energy) diets for 6-29 weeks. Our initial studies assessed the immunoreactive levels of PKC alpha, beta, gamma, delta, epsilon and zeta and PKC alpha beta gamma using an antibody to a shared epitope. We detected PKC alpha, epsilon, delta and zeta in sufficient quantity for dietary studies. Dietary fat and energy did not significantly modify the presence of PKC epsilon or delta. We observed a 30% and 40% reduction in PKC alpha in comparison with control diet in cytosolic and particulate fractions respectively, from mice pre-fed ER diet. Reductions of 72% and 82% in cytosolic and particulate PKC were observed respectively with the alpha beta gamma antibody. ER diet reduced the overall amount of PKC zeta in cytosol and particulate by 42% and 59% respectively, with the cytosolic reduction being greater in mice pre-fed the restricted diets for 21-29 weeks. HF diet did not modify the protein levels of the PKC isoenzymes studied. The level of phorbol-12,13-dibutyrate binding to epidermal cells was assessed to determine if the reduction in PKC protein in the epidermis of ER mice would result in altered phorbol binding. Phorbol binding in cells isolated from mice fed ER diet was reduced in mice pre-fed the ER diet for 20-22 weeks in comparison with binding to cells from mice fed control diet. These results suggest that ER diet reduces specific PKC isoenzymes and the binding of phorbol esters in epidermis of mice. These observations may account for the inhibition of phorbol ester promotion of skin tumors in ER mice.

Previous studies in our laboratory have shown that promotion of two-stage skin carcinogenesis in the SENCAR mouse model was inhibited in mice fed energy-restricted/low-fat diets, and elevated in mice fed high-fat diets. Studies reported here describe the influence of dietary energy restriction from fat and carbohydrate (ER) or high-fat (HF) diet on early promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) and on late promotion by mezerein (MEZ). Female SENCAR mice were initiated topically with 10 nmol 7,12-dimethylbenz[a]anthracene (DMBA) in 0.2 ml acetone at 9 weeks of age. For the following 2 weeks they received 3.2 nmol TPA in 0.2 ml acetone twice weekly, and for the next 16 weeks they received 10 nmol MEZ in 0.2 ml acetone twice weekly. All mice were fed control diet before TPA began and following the final MEZ treatment. Control mice received the control diet (c) throughout TPA and MEZ (C/C). The six experimental groups received: (1) ER diet throughout TPA and MEZ treatment (ER/ER); (2) HF diet throughout TPA and MEZ treatment (HF/HF); (3) ER during TPA (ER/C); (4) ER during MEZ (C/ER); (5) HF diet during TPA (HF/C); or (6) HF diet during MEZ (C/HF). Papilloma incidence and multiplicity, and carcinoma incidence were similarly reduced in the mice fed ER diet during MEZ (ER/ER and C/ER groups). In comparing the HF groups, papilloma multiplicity was highest in the HF/C group, intermediate in the C/C and lowest in the C/HF groups, but papilloma and carcinoma incidences were not modified by the HF diet protocols. Papilloma regression was greater in the C/HF group (27%, 4 regressions/15 tumor-bearing mice) than in the controls (0/18) during weeks 21-23, immediately following the end of MEZ treatment (P < 0.05).

Exposure of mammalian cells to UV radiation and other DNA-damaging agents triggers a response known as the UV response. This induction response involves a large number of genes including c-jun, cell-cycle regulatory proteins, specific repair enzymes, and the tumor suppressor gene p53. Altered expression of these genes following DNA damage is hypothesized to result in G1 arrest, thereby allowing cells to repair DNA damage prior to cell division. In the present study, we investigated expression of the p53 gene in mouse keratinocyte cell line 308 after exposure to UV-B light at a biologically relevant dose. Irradiation of 308 cells with 40 J/m2 UV-B resulted in a 4- to 10-fold induction in the level of p53 protein, peaking at 5 h post-irradiation. Northern blot analysis of RNA from UV-B irradiated cells showed no change in the steady-state level of p53 mRNA following irradiation. However, the half-life of p53 protein in UV-B irradiated 308 cells was extended approximately 7-fold, from 30 to 200 min. Additional studies were performed with specific anti-p53 monoclonal antibodies to establish whether UV-B irradiation induced a conformational change in p53 protein in irradiated cells. Metabolic labeling with 35S-methionine followed by immunoprecipitation with p53 monoclonal antibody PAb246, which recognizes the wild-type murine p53 protein, demonstrated that the p53 protein present in 308 cells possessed the wild-type conformation both before and after UV-B irradiation. In contrast, p53 antibody PAb240, which recognizes a 'conformation-dependent' epitope, was not reactive with the p53 protein present in 308 cells. Therefore, we conclude that the induction of p53 protein in mouse keratinocytes following UV-B irradiation occurred posttranscriptionally, and was due to a significant increase in p53 protein half-life.

NIH3T3 cells transfected with an activated Ha-ras oncogene were treated with L-PHA, the leukoagglutinin from red kidney beans. Cell lines resistant to L-PHA-mediated cytotoxicity were isolated and found to contain reduced levels of L-PHA-binding oligosaccharides. The levels of N-acetylglucosaminyltransferase V, the enzyme responsible for the initiation of the beta 1-6 branch, were reduced in L-PHA-resistant cells. Tumorigenicity in nude mice was unchanged by the change in oligosaccharide expression, but the ability to form lung tumors after intravenous injection was significantly reduced. These results demonstrate that the ability of NIH3T3 cells transfected with an activated Ha-ras oncogene to form lung tumors after intravenous injection into nude mice is reduced in all six L-PHA selected cell lines containing a reduction in beta 1-6 branched Asn-linked oligosaccharides.

Male F344 rats were fed N[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) for up to 4 wk, then given the basal diet with or without 5% sodium saccharin for up to 100 wk. In a previous study, we demonstrated point mutations in codons 12 and 61 of Ha-ras gene among eleven transitional cell carcinomas (TCC), one undifferentiated carcinoma, and two sarcomas of the urinary bladder (Mol Carcinogen 3:210-215, 1990). In this study, Ha-ras, Ki-ras, and N-ras sequences were examined by polymerase chain reaction (PCR) and direct DNA sequencing. The results confirm the point mutation in codon 61 (CAA to CGA in 5 TCCs and to CTA in one TCC) of the Ha-ras gene. Mutation at codon 12 was not confirmed. No mutation was found in the Ki-ras gene. Sequences of the N-ras gene exons 1 and 2 were determined, and no mutations was detected. These results suggest the involvement of activated Ha-ras gene, but not Ki-N or N-ras gene, in rat urinary bladder carcinogenesis induced by FANFT. Subsequent sodium saccharin administration did not affect the changes in Ha-ras gene.

Studies reported here determined the effect of dietary fat level on membrane phospholipid composition, phosphoinositide labeling, 1,2-sn-diacylglycerol and protein kinase C activity in epidermal cells from female Sencar mice. Animals were fed either high fat (24.6 g/100 g diet) or control (5 g/100 g diet) diets at constant energy intake for 6 to 7 wk or 15 to 22 wk, and epidermal cells were isolated. The level of phosphatidylinositol was significantly lower in the animals fed the control diet than in the animals fed the high fat diet (0.6 vs. 1.2 nmol/10(6) cells). The fatty acid composition of the phospholipids showed significantly lower arachidonic acid level in phosphatidylinositol when the animals were fed the high fat diet. Protein kinase C activity in the solubilized particulate and soluble fraction of the cells was 131 +/- 18% and 62 +/- 14% greater, respectively, in animals fed the high fat diet compared with animals fed control diet. The level of 1,2-sn-diacylglycerol was significantly higher in animals fed the high fat diet (mean nmol/mg lipid +/- SEM: control, 4.5 +/- 0.5; high fat, 7.0 +/- 0.5). Incorporation of [3H]inositol into inositol lipid was not altered by diet. Because protein kinase C and 1,2-sn-diacylglycerol have been implicated in tumor promotion, the increase in protein kinase C activity and the elevation of 1,2-sn-diacylglycerol in cells from animals fed the high fat diet may be important in the high cancer rate observed with these diets.

The Ha-ras oncogene has been shown to be point-mutated and overexpressed in papillomas induced by the two-stage skin tumorigenesis regimen of 7,12-dimethylbenz[a]anthracene (DMBA) and promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). Glucocorticoids inhibit mouse skin tumorigenesis when applied with the initiation agent or with the promoting agent. We have extended these studies to evaluate whether dexamethasone (Dex) treatment could inhibit development of already established tumors. An additional objective of this study was to investigate whether glucocorticoids directly inhibit Ha-ras gene expression at the level of transcription during skin tumorigenesis. In this report we demonstrate that topical Dex treatments significantly suppressed the formation of additional tumors relative to the acetone control group. However, Northern blot analysis of total RNA isolated from representative tumors during a series of sequential weeks of promotion indicated that Dex did not have a direct effect on Ha-ras steady-state mRNA levels despite the decrease in additional tumor numbers in the Dex-treated groups. We also investigated short-term effects of Dex on endogenous Ha-ras expression in normal mouse epidermis. Topical Dex administration had no effect on endogenous Ha-ras steady-state mRNA levels in normal skin after 2 or 24 h. To ensure that endogenous corticosterone levels in the SENCAR mouse were not influencing our results, Ha-ras mRNA levels in epidermis from SENCAR mice adrenalectomized 48 h prior to being killed were compared to Ha-ras levels in normal epidermis by Northern blot analysis. The data from this analysis revealed that bilateral adrenalectomy had no effect on Ha-ras steady-state mRNA levels in epidermis compared to ras levels in normal mouse epidermis. In summary, our results demonstrate that although Dex can inhibit further tumor development in DMBA/TPA-treated mouse epidermis, it does not do so by directly effecting Ha-ras gene expression in mouse epidermis.