Transformation Of JB6 Research Articles

Abstract 5292: Visceral adiposity-stimulated genotoxicity and malignant transformation of epithelial cells

Abstract Obesity is a global epidemic with a predicted rate of 42% in the U.S.A by 2050. Epidemiologic studies show that obesity is a risk factor for developing cancer; however, the molecular mechanism has not been fully elucidated. Our published data demonstrate that fibroblast growth factor-2 (FGF2) released from fat cells (adipocytes) in the visceral adipose tissue (VAT) induces transformation/tumorigenicity in the skin and mammary epithelial cells. Specifically, FGF2 released from VAT stimulates epithelial cell growth in soft agar by inducing the proto-oncogene c-Myc. Growth in soft agar is a measure of transformation/tumorigenicity; neither transformation nor c-Myc induction in epithelial cells was reversible. c-Myc overexpression can initiate a process of genetic instability linked to tumor initiation. Our discovery of this novel direct path of VAT-stimulated tumorigenesis adds mechanistic insight to our earlier discovery that VAT secretions promote UVR-induced nonmelamoma skin cancer. The objective of our current study was to determine the mechanism by which FGF2 stimulates malignant transformation. We hypothesized that FGF2 from VAT induces c-Myc and subsequent genomic instability in epithelial cells, leading to increased carcinogenesis. To test hypothesis we generated a filtered conditioned-medium from the human VAT treated MCF-10A (mammary epithelial) and JB6 P+ (skin epithelial) cells and measured several downstream mediators of FGF2 and activation of FGFR-1 (FGF2 receptor). Following VAT treatment, epithelial cells demonstrated induced c-Myc protein expression along with ROS accumulation, elevated γ-H2AX foci, and increased micronucleus (MN) formation. We found that inhibition of c-Myc attenuated VAT-induced neoplastic transformation of MCF-10A and JB6 P+ cells, while constitutive activation of the c-Myc-induced spontaneous neoplastic transformation of JB6 P+ cells. Collectively, our data suggested FGF2 released from VAT interacts with FGFR-1 and activates c-Myc. The role of c-Myc in the formation of MN and DNA damage is under investigation. Determining the impact of excess VAT on cancer will lead to strategies to help prevent adiposity-associated cancers and identify individuals at risk for disease or individuals who may be susceptible to compounded genotoxicity due to DNA-damaging environmental exposures. Citation Format: Debrup Chakraborty, Vladislav Jdanov, Vanessa Benham, Blair Bullard, Jamie J. Bernard. Visceral adiposity-stimulated genotoxicity and malignant transformation of epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5292.

Veratramine modulates AP-1-dependent gene transcription by directly binding to programmable DNA.

Because the transcription factor activator protein-1 (AP-1) regulates a variety of protein-encoding genes, it is a participant in many cellular functions, including proliferation, transformation, epithelial mesenchymal transition (EMT), and apoptosis. Inhibitors targeting AP-1 have potential use in the treatment of cancer and other inflammatory diseases. Here, we identify veratramine as a potent natural modulator of AP-1, which selectively binds to a specific site (TRE 5′-TGACTCA-3′) of the AP-1 target DNA sequence and regulates AP-1-dependent gene transcription without interfering with cystosolic signaling cascades that might lead to AP-1 activation. Moreover, RNA-seq experiments demonstrate that veratramine does not act on the Hedgehog signaling pathway in contrast to its analogue, cyclopamine, and likely does not harbor the same teratogenicity and toxicity. Additionally, veratramine effectively suppresses EGF-induced AP-1 transactivation and transformation of JB6 P+ cells. Finally, we demonstrate that veratramine inhibits solar-ultraviolet-induced AP-1 activation in mice. The identification of veratramine and new findings in its specific regulation of AP-1 down stream genes pave ways to discovering and designing regulators to regulate transcription factor.

The β‐blockers carvedilol and nebivolol inhibit neoplastic transformation through a β2‐adrenergic receptor independent mechanism

β‐adrenergic receptors (β‐ARs) are crucial mediators of physiological responses to catecholamines (i.e. epinephrine and norepinephrine), and β‐blockers competitively inhibiting β‐ARs thereby preventing the associated cardiovascular effects of β‐ARs. Aside from their cardiovascular function, β‐blockers are potential chemopreventive agents. Previous studies identified a subset of β‐blockers as potential cancer preventive agents. Carvedilol and nebivolol inhibit EGF‐induced neoplastic transformation of JB6 P+ cells, a non‐cancerous but transformation sensitive murine epidermal cell line. However, the antineoplastic mechanism(s) of action have yet to be elucidated. Therefore, we sought to evaluate the role of the β2‐AR, the only β‐AR present in JB6 P+ cells, in the β‐blocker antineoplastic effect. First, an EGF‐mediated soft agar transformation assay was utilized to evaluate the combined effect of carvedilol with either CGP 12177 or nadolol; CGP 12177 and nadolol lack a cancer preventive effect. 10 μM CGP 12177 (KD= −9.39) had no effect on carvedilol (KD= −9.40) mediated inhibition of EGF‐induced transformation of JB6 P+ cells: −6.48±0.09 IC50 versus −6.67±0.05 IC50 for carvedilol and carvedilol plus CGP 12177, respectively. Similarly, carvedilol combined with 10 μM nadolol (KD= −8.60) resulted in no shift in the concentration‐response curve (−6.55±0.03 IC50 versus −6.50±0.06 IC50). Since carvedilol is a non‐selective β‐AR and α1‐AR antagonist, we assessed the involvement of the α1‐AR by combining the β‐blocker nebivolol, that lacks α1‐AR activity, with 10 μM CGP 12177. Combination of nebivolol (KD= −7.91 for the β2‐AR) and CGP 12177 did not alter the potency of nebivolol (−6.46±0.11 and −6.81±0.12 for nebivolol and nebivolol plus CGP 12177, respectively). To further the pharmacological studies, the efficacy and potency of carvedilol in preventing EGF‐induced transformation of JB6 P+ cells was compared in scramble control shRNA and Adrb2 (β2‐AR) shRNA infected cells. With 72% β2‐AR knockdown, carvedilol prevented EGF‐mediated transformation of JB6 P+ cells (IC50= −6.02±0.07), although with a slightly reduced potency compared to the scramble control (IC50= −6.24±0.06). The data demonstrate that the β‐blocker carvedilol and likely nebivolol function as a cancer preventive agent independently of their widely‐understood action as β‐AR blockers.

Abstract 2904: Induction of autophagy reveals a cytoprotective mechanism by which DNA polymerase gamma (pol-γ) prevents UVB-induced skin cancer

Abstract DNA polymerase γ (polγ), a major mitochondrial DNA repair enzyme, is essential for replication and repair of the mitochondrial genome. It is well-established that mitochondrial genomic stability is important for healthy cell survival; however, whether and how polγ plays a role in autophagic response remain unknown. Our previous studies demonstrated that polγ is inactivated upon exposure to ultraviolet B (UVB) irradiation by a peroxynitrite-mediated mechanism. In this study, we identified tyrosine 964, which is near the active site of polγ, as the target of nitration-induced inactivation by the use of LC/MS/MS mass spectroscopy coupled to immunoprecipitation, Western blotting and a 3` to 5` exonuclease activity assay. Treatment of mouse skin epithelial cells with UVB radiation validated the presence of polγ nitration and reduction of its enzymatic activity in vivo. To determine the effect of UVB in autophagic responses, mouse skin epithelial (JB6) cells were transfected with GFP-labeled LC-3 cDNA and exposed to UVB radiation, or wild-type C57BL/6 mice were exposed to UVB radiation and examined for LC-3 cleavage products. UVB radiation of JB6 cells and mouse skin increased LC-3 cleavage products and beclin levels, suggesting increased autophagic response to UVB treatment both in vitro and in vivo. To elucidate the role of Polγ in autophagic responses, the expression of polγ in JB6 cells was inhibited by siRNA for polγ. Consistent with the role of polγ in preventing UVB-induced autophagy, knockdown of polγ led to an increase LC-3 cleavage products. Knockdown of polγ induced prosurvival autophagic responses via the mTOR-signaling-complex-1(mTORC1) and complex-2(mTORC2). Interestingly, the mTORC2 complex plays a prominent role in the prosurvival autophagy responses based on the bromodeoxyuridine uptake assay, following knockdown and overexpression approaches. Importantly, suppression of polγ alone leads to transformation and invasion ofJB6 cells, which are increased following UVB treatment. The results reveal a unique role of Polγ in autophagy and support the role of Polγ in the suppression of prosurvival autophagic response to UVB- mediated carcinogenesis. Citation Format: Sanjit K. Dhar, Vasu Bakthavachalu, Tadahide Izumi, Jing Chen, Haining Zhu, Daret K. St. Clair. Induction of autophagy reveals a cytoprotective mechanism by which DNA polymerase gamma (pol-γ) prevents UVB-induced skin cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2904. doi:10.1158/1538-7445.AM2015-2904

3,4-Dihydroxytoluene의 Raf-1 신호전달체계 억제를 통한 암예방 효능

Summary In summary, the rutin metabolite DHT significantly reduced EGF-induced neoplastic transformation in JB6 P+ cells. This inhibition was mediated mainly by blocking the Raf/MEK/ERK signaling pathway and subsequent suppression of AP-1 activities. DHT attenuated Raf-1 kinase activity by directly binding to Raf-1 in vitro and ex vivo. Taken together, these results suggest that Raf-1 may be a critical molecular target to suppress DHT- induced neoplastic transformation, which is mainly attributable to the chemopreventive potential of several foods containing rutin. Acknowledgments This research was supported by the Kyungpook National University Research Fund, 2009. References Alonso-Castro AJ, Dominguez F, Garcia-Carranca A. 2013. Rutin exerts antitumor effects on nude mice bearing SW480 tumor. Arch Med Res 44:346-351 Antonyak MA, Moscatello DK, Wong AJ. 1998. Constitutive activation of c-Jun N-terminal kinase by a mutant epidermal growth factor receptor. J Biol Chem 273:2817-2822Bode AM, Dong Z. 2005. Signal transduction pathways in cancer development and as targets for cancer prevention. Prog Nucleic Acid Res Mol Biol 79:237-297 Cheepala SB, Yin W, Syed Z, Gill JN, McMillian A, Kleiner HE, Lynch M, Loganantharaj R, Trutschl M, Cvek U, Clifford JL. 2009. Identification of the B-Raf/Mek/Erk MAP kinase pathway as a target for all-trans retinoic acid during skin cancer promotion. Mol Cancer 8:27 Choi KS, Kundu JK, Chun KS, Na HK, Surh YJ. 2014. Rutin inhibits UVB radiation-induced expression of COX-2 and iNOS in hairless mouse skin: p38 MAP kinase and JNK as potential targets. Arch Biochem Biophys 559:38-45.Dong Z, Birrer MJ, Watts RG, Matrisian LM, Colburn NH. 1994. Blocking of tumor promoter-induced AP-1 activity inhibits induced transformation in JB6 mouse epidermal cells. Proc Natl Acad Sci USA 91:609-613Garcia-Lafuente A, Guillamon E, Villares A, Rostagno MA, Martinez JA. 2009. Flavonoids as anti-inflammatory agents: implications in cancer and cardiovascular disease. Inflamm Res 58:537-552 Glasser G, Graefe EU, Struck F, Veit M, Gebhardt R. 2002. Comparison of antioxidative capacities and inhibitory effects on cholesterol biosynthesis of quercetin and potential meta-bolites. Phytomedicine 9:33-40 He Z, Cho YY, Liu G, Ma WY, Bode AM, Dong Z. 2003. p38 Mitogen-activated protein kinase regulation of JB6 Cl41 cell transformation promoted by epidermal growth factor. J Biol Chem 278:26435-26442 Huang C, Ma WY, Dawson MI, Rincon M, Flavell RA, Dong Z. 1997. Blocking activator protein-1 activity, but not acti-vating retinoic acid response element, is required for the antitumor promotion effect of retinoic acid. Proc Natl Acad Sci USA 94:5826-5830Huang CS, Ma WY, Young MR, Colburn N, Dong ZG. 1998. Shortage of mitogen-activated protein kinase is responsible for resistance to AP-1 transactivation and transformation in mouse JB6 cells. Proc Natl Acad Sci USA 95:156-161Ito S, Inoue S, Yamamoto Y, Fujita K. 1981. Synthesis and antitumor activity of cysteinyl-3,4-dihydroxyphenylalanines

Abstract 1256: The chemopreventive effects of carvedilol on skin carcinogenesis

Abstract Skin cancer, the majority of which is non-melanoma skin cancer (NMSC), is the most common type of cancer in the US. The rate of NMSC is increasing mainly due to depletion of the ozone layer and increased exposure to ultraviolet radiation. To reduce the risk of skin cancer, it is recommended to all individuals to limit sun exposure and use sunscreens. Despite these efforts, the incidence of NMSC continues to rise. Novel chemopreventive targets and nontoxic agents are needed. Epidemiological studies identified an association of psychosocial factors such as chronic stress or depression with cancer onset and progression. These factors are partly mediated by activation of the sympathetic nervous system which results in release of norepinephrine and epinephrine, the effects of which are mediated through the alpha- and beta-adrenergic receptors (alpha- and beta-AR). Further, the use of beta-AR antagonists (beta-blockers) has been associated with reduced cancer incidence. However, whether beta-blockers have chemopreventive activity or whether beta-ARs contribute to carcinogenesis is unknown. To determine whether agonizing or antagonizing beta-ARs affect skin cell transformation, we tested the beta-AR agonist isoproterenol (Iso) and antagonist carvedilol on epidermal growth factor (EGF)-mediated transformation of JB6 P+ cells (a skin cell model to study tumor promotion). The cells were treated with EGF (10 ng/mL) plus 0.1 µM or 1.0 µM Iso, yet no effect was observed. However, treatment with carvedilol dose-dependently inhibited the formation of EGF-induced soft agar colonies. Such effect was not caused by growth inhibition, because cytotoxicity and inhibition of cell proliferation were not observed for tested concentrations. Next, the expression of beta1-, 2- and 3-AR was determined in JB6 cells using RT-PCR. The results showed that only beta2-AR was detectable. We also determined whether beta-ARs are expressed in other types of cancer. In tissues derived from a rat model of 7, 12-dimethylbenz[α]anthracene (DMBA)-induced mammary cancer, expression of beta1- and 2-AR was up-regulated in tumors in comparison with normal tissues. The human cancer cell lines A549 and MDA-MB-231 also express beta2-AR. To determine the in vivo chemopreventive activity of carvedilol, we used a skin hyperplasia model in SENCAR mice induced by topical treatment with 100 nM DMBA twice a week for four weeks. Carvedilol was tested topically (5 and 10 µM) or orally (5 and 20 mg/kg), beginning at two weeks before the first dose of DMBA, three times a week, for six weeks. DMBA alone increased the epidermal thickness from the average of 77.8 + 13.6 nm in normal skin to 294 + 49.7 nm in DMBA-treated skin. Both topical and oral carvedilol treatment inhibited DMBA-induced hyperplasia (P < 0.05). In conclusion, these results suggest that beta-blockers may have chemopreventive activity. This study also suggests that beta-ARs may serve as a novel target for cancer prevention. Citation Format: Andy Chang, Mandy Liu, Steven Yeung, Jijun Hao, Cyrus Parsa, Robert Orlando, Bradley Andresen, Ying Huang. The chemopreventive effects of carvedilol on skin carcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1256. doi:10.1158/1538-7445.AM2014-1256

Abstract 2589: The P110 subunit of PI3-K is a therapeutic target of acacetin in skin cancer.

Abstract Verifying potential target proteins of phytochemicals is important for explaining their health benefits. In the present study, we examined the chemopreventive effect and molecular target of acacetin, which is a flavonoid from Robinia Psuedoacacia also known as Black Locust. Treatment with acacetin significantly inhibits EGF-induced transformation of JB6 P+ cells. Western blot assay results revealed that acacetin suppresses EGF-induced phosphorylation of Akt/p70S6K, which is a well-known downstream signaling molecule of phosphatidylinositol 3-kinase (PI3-K). In vitro kinase and pull-down assay results showed that acacetin significantly inhibits PI3-K activity by direct binding. Additionally, acacetin also attenuated EGF-induced PI3-K activity and binds with PI3-K in JB6 P+ cells. Acacetin showed significant effects against anchorage-dependent and -independent cell growth in cells displaying higher PI3-K activity compared with those exhibiting lower PI3-K activity. Binding assay data and computational modeling showed that acacetin binds in an ATP-competitive manner with the p110 kinase domain of PI3-K and interacts with Val828, Glu826 Asp911 Trp760, Ile777, Ile825, Tyr813, Ile910 and Met900. Acacetin reduced SK-MEL-28 xenograft tumor growth and phosphorylation of Akt in vivo. Overall, these results indicate that acacetin is an ATP competitive PI3-K inhibitor and might be a promising chemopreventive agent targeting PI3-K activity. Citation Format: Jon Eun Kim, Sung Keun Jung, Sanguine Byun, Sung-Young Lee, Myoung Ok Kim, Tae Gyu Lim, Ann M. Bode, Hyong Joo Lee, Ki Won Lee, Zigang Dong. The P110 subunit of PI3-K is a therapeutic target of acacetin in skin cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2589. doi:10.1158/1538-7445.AM2013-2589

Norathyriol suppresses transformation in JB6 P+ cells by the inhibition of Akt

Chemoprevention has been acknowledged as an important and practical strategy for the management of skin cancer. Norathyriol, a naturally occurring compound present in various plants, has a potent anticancer-promoting activity. The aim was to investigate the chemopreventive activity of norathyriol on JB6 P+ cells. A soft agar assay was used to detect the effect of norathyriol on cell transformation. The activator protein-1 (AP-1) transactivation activity was examined by the luciferase assay. Norathyriol inhibited epidermal growth factor (EGF)- and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic cell transformation in a dose-dependent manner. The activation of activator protein-1 was dose dependently suppressed by norathyriol treatment. Western blot data revealed that norathyriol attenuated the phosphorylation of Akt. Norathyriol exerts a potent chemopreventive activity by inhibiting Akt activation in neoplastic cell transformation.

The chemopreventive effects of Protandim: modulation of p53 mitochondrial translocation and apoptosis during skin carcinogenesis.

Protandim, a well defined dietary combination of 5 well-established medicinal plants, is known to induce endogenous antioxidant enzymes, such as manganese superoxide dismutase (MnSOD). Our previous studies have shown through the induction of various antioxidant enzymes, products of oxidative damage can be decreased. In addition, we have shown that tumor multiplicity and incidence can be decreased through the dietary administration of Protandim in the two-stage skin carcinogenesis mouse model. It has been demonstrated that cell proliferation is accommodated by cell death during DMBA/TPA treatment in the two-stage skin carcinogenesis model. Therefore, we investigated the effects of the Protandim diet on apoptosis; and proposed a novel mechanism of chemoprevention utilized by the Protandim dietary combination. Interestingly, Protandim suppressed DMBA/TPA induced cutaneous apoptosis. Recently, more attention has been focused on transcription-independent mechanisms of the tumor suppressor, p53, that mediate apoptosis. It is known that cytoplasmic p53 rapidly translocates to the mitochondria in response to pro-apoptotic stress. Our results showed that Protandim suppressed the mitochondrial translocation of p53 and mitochondrial outer membrane proteins such as Bax. We examined the levels of p53 and MnSOD expression/activity in murine skin JB6 promotion sensitive (P+) and promotion-resistant (P-) epidermal cells. Interestingly, p53 was induced only in P+ cells, not P- cells; whereas MnSOD is highly expressed in P- cells when compared to P+ cells. In addition, wild-type p53 was transfected into JB6 P- cells. We found that the introduction of wild-type p53 promoted transformation in JB6 P- cells. Our results suggest that suppression of p53 and induction of MnSOD may play an important role in the tumor suppressive activity of Protandim.

7,3′,4′-Trihydroxyisoflavone Inhibits Epidermal Growth Factor-induced Proliferation and Transformation of JB6 P+ Mouse Epidermal Cells by Suppressing Cyclin-dependent Kinases and Phosphatidylinositol 3-Kinase

Numerous in vitro and in vivo studies have shown that isoflavones exhibit anti-proliferative activity against epidermal growth factor (EGF) receptor-positive malignancies of the breast, colon, skin, and prostate. 7,3',4'-Trihydroxyisoflavone (7,3',4'-THIF) is one of the metabolites of daidzein, a well known soy isoflavone, but its chemopreventive activity and the underlying molecular mechanisms are poorly understood. In this study, 7,3',4'-THIF prevented EGF-induced neoplastic transformation and proliferation of JB6 P+ mouse epidermal cells. It significantly blocked cell cycle progression of EGF-stimulated cells at the G(1) phase. As shown by Western blot, 7,3',4'-THIF suppressed the phosphorylation of retinoblastoma protein at Ser-795 and Ser-807/Ser-811, which are the specific sites of phosphorylation by cyclin-dependent kinase (CDK) 4. It also inhibited the expression of G(1) phase-regulatory proteins, including cyclin D1, CDK4, cyclin E, and CDK2. In addition to regulating the expression of cell cycle-regulatory proteins, 7,3',4'-THIF bound to CDK4 and CDK2 and strongly inhibited their kinase activities. It also bound to phosphatidylinositol 3-kinase (PI3K), strongly inhibiting its kinase activity and thereby suppressing the Akt/GSK-3beta/AP-1 pathway and subsequently attenuating the expression of cyclin D1. Collectively, these results suggest that CDKs and PI3K are the primary molecular targets of 7,3',4'-THIF in the suppression of EGF-induced cell proliferation. These insights into the biological actions of 7,3',4'-THIF provide a molecular basis for the possible development of new chemoprotective agents.

Inhibition of AP-1 and MAPK signaling and activation of Nrf2/ARE pathway by quercitrin

Quercitrin, glycosylated form of flavonoid compounds, is widely distributed in nature. Extensive studies have demonstrated that quercitrin exhibits strong antioxidant and anti-carcinogenic activities. However, the molecular mechanism is poorly understood. The present study examines the effects of quercitrin on tumor promotion in mouse JB6 cells, a validated model for screening cancer chemopreventive agents and elucidating the molecular mechanisms. Quercitrin blocked TPA-induced neoplastic transformation in JB6 P+ cells. Pretreatment of JB6 cells with quercitrin down-regulated transactivation of AP-1 and NF-kappaB induced by UVB or TPA. In the skin of AP-1-luciferase transgenic mice, topical treatment of the mouse with quercitrin markedly blocked the TPA-induced AP-1 transactivation. Further studies indicated that these inhibitory actions appear to be mediated through the inhibition of MAPKs phosphorylation, including ERKs, p38 kinase, and JNKs. In addition, quercitrin stimulated the activation of NF-E2-related factor (Nrf2) and GST ARE-luciferase activity. Comet assays showed that quercitrin could block DNA damage induced by UVB. To our knowledge, these results provide the first evidence that quercitrin contributes to the inhibition of neoplastic transformation by blocking activation of the MAPK pathway and stimulation of cellular protection signaling. Moreover, to our knowledge, these findings provide the first molecular basis for the anti-carcinogenic action of quercitrin.

The resveratrol analogue 3,5,3′,4′,5′‐pentahydroxy‐trans‐stilbene inhibits cell transformation via MEK

Resveratrol, present in grapes and red wine, is reported to be a natural chemopreventive agent against cancer. However, the concentrations required to exert these effects may be difficult to achieve by drinking only 1 or 2 glasses of red wine a day. Therefore, developing more potent, nontoxic analogues of resveratrol may provide a feasible means of achieving an effective physiologic concentration. Here we report that the resveratrol analogue, 3,5,3',4',5'-pentahydroxy-trans-stilbene (RSVL2), inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation in JB6 P+ mouse epidermal cells. Further, we identified MEK/ERK signaling as the direct molecular target for the anticancer effects of RSVL2 and demonstrated that RSVL2 inhibited MEK1, but not Raf1 or ERK2 kinase activity. RSVL2 also dose-dependently suppressed MEK1 kinase activity induced by TPA and the inhibition of H-Ras-induced cell transformation was much stronger for RSVL2 than for PD098059 or resveratrol. Both in vitro and ex vivo pull-down assays indicated that RSVL2, but not resveratrol, directly bound with GST-MEK1, but did not compete with ATP for binding. Docking data indicated that the low inhibitory activity of resveratrol might be due to the lack of the hydroxyl group at the meta position of the B ring, thereby preventing resveratrol from forming a hydrogen bond with the backbone amide group of Ser212, which is the key interaction for stabilizing the inactive conformation of the activation loop.

Inhibitory effects of caffeine analogues on neoplastic transformation: structure-activity relationship

Some xanthine analogues, including 1,3,7-trimethylxanthine (caffeine) and 1,3-dimethylxanthine (theophylline), have been shown to exert anticancer activities in both cell culture and animal models. The present study focused on the relationship of structure and activity of 50 different caffeine analogues in preventing epidermal growth factor (EGF)-induced malignant transformation of mouse epidermal JB6 promotion-sensitive (P+) Cl41 (JB6 P+) cells. Results indicated that the inhibition of cell transformation by the 1,3,7-trialkylxanthines depends on the number of carbons at the alkyl groups R1 and R3, but not R7. Notably, 1-ethyl-3-hexylxanthine (xanthine 70) was the most effective compound for inhibiting EGF-induced neoplastic transformation among the 50 xanthine analogues tested. The 50% inhibition of cell transformation (ICT(50)) value for xanthine 70 was 48- or 75-fold less than the ICT(50) value of caffeine or theophylline, respectively. Further study revealed that xanthine 70 (5-40 muM) dose dependently inhibited EGF-induced transactivation of activator protein 1 (AP-1), whereas theophylline or caffeine (up to 500 muM) had no effect on AP-1 activity. In addition, xanthine 70 (10 muM) inhibited 12-O-tetradecanoylphorbol-13-acetate- or H-Ras-induced neoplastic transformation in JB6 P+ cells by 78.2 or 62.0%, respectively. Collectively, these results indicated that the number of carbons at R1 and R3 is important for the antitumor-promoting activity of the trialkylxanthines and xanthine 70 might be a promising anticancer agent.

Myricetin is a novel natural inhibitor of neoplastic cell transformation and MEK1

Evidence suggests that mitogen-activated protein kinase kinase (MEK) plays a role in cell transformation and tumor development and might be a significant target for chemoprevention. 3,5,4'-Trihydroxy-trans-stilbene (resveratrol), a non-flavonoid polyphenol found in various foods and beverages, including red wines, is reported to be a natural chemopreventive agent. However, the concentrations required to exert these effects might be difficult to achieve by drinking only one or two glasses of red wine a day. On the other hand, the flavonol content of red wine is approximately 30 times higher than that of resveratrol. Here we demonstrated that 3,3',4',5,5',7-hexahydroxyflavone (myricetin), one of the major flavonols in red wine, is a novel inhibitor of MEK1 activity and transformation of JB6 P+ mouse epidermal cells. Myricetin (10 microM) inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA) or epidermal growth factor (EGF)-induced cell transformation by 76 or 72%, respectively, compared with respective reductions of 26 or 19% by resveratrol (20 microM). A combination of myricetin and resveratrol exerted additive but not synergistic effects on either TPA- or EGF-induced transformation. Myricetin, but not resveratrol, attenuated tumor promoter-induced activation of c-fos or activator protein-1. Myricetin strongly inhibited MEK1 kinase activity and suppressed TPA- or EGF-induced phosphorylation of extracellular signal-regulated kinase (ERK) or p90 ribosomal S6 kinase, downstream targets of MEK. Moreover, myricetin inhibited H-Ras-induced cell transformation more effectively than either PD098059, a MEK inhibitor, or resveratrol. Myricetin directly bound with glutathione S-transferase-MEK1 but did not compete with ATP. Overall, these results indicated that myricetin has potent anticancer-promoting activity and mainly targets MEK signaling, which may contribute to the chemopreventive potential of several foods including red wines.

Effects of polyphenols derived from fruit of Crataegus pinnatifida on cell transformation, dermal edema and skin tumor formation by phorbol ester application

The dried fruits of Crataegus pinnatifida have been used traditionally as oriental medicine and local soft drink material recently. Previously, we demonstrated that C. pinnatifida exhibited anti-oxidation and anti-inflammatory potential. To clarify the active components in anti-transformation and anti-tumor promotion, we collected the polyphenol fraction (CF-TP) of hot-water extracts from dried fruits of C. pinnatifida for the following study. By anchorage-independent transformation assay, CF-TP significantly inhibited 12- O-tetradecanoylphorbol-13-acetate (TPA)-induced cell transformation in JB6 P + cells. Moreover, we found that CF-TP inhibited the expression of osteopontin (OPN), a transformational marker, and the activation of NF-κB and AP-1 induced by TPA in JB6 P + cells. In addition, we evaluated the effect of CF-TP on TPA application to ICR mouse skin with measurement of H 2O 2 production, myeloperoxidase (MPO) activity, edema formation, epidermal thickness and leukocyte infiltration. As a result, CF-TP significantly inhibited the generation of reactive oxygen species (ROS) and the phenomena of inflammation induced by TPA. It also suppressed the expression of COX-2 and iNOS, and the activation of ornithine decarboxylase (ODC). Furthermore, CF-TP inhibited benzo[ a]pyrene (B[ a]P)/TPA-induced skin tumor formation and decreased the incidence of tumor. These results indicate that CF-TP possesses potential as a cancer chemopreventive agent against tumor promotion.

Peonidin Inhibits Phorbol‐Ester–Induced COX‐2 Expression and Transformation in JB6 P+ Cells by Blocking Phosphorylation of ERK‐1 and ‐2

Abnormal upregulation of cyclooxygenase-2 (COX-2) has been frequently observed in various types of transformed and cancerous cells. Numerous anti-inflammatory agents have been shown to exert chemopreventive effects by targeting COX-2, a rate-limiting enzyme involved in the inflammatory process. Anthocyanins are naturally occurring polyphenolic compounds that endow various fruits, vegetables, and plants with intense colors. Peonidin is another representative anthocyanidin, but its chemopreventive potential has not been fully described. This article investigated the effect of peonidin on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced COX-2 expression and transformation in JB6 P(+) mouse epidermal cells (JB6 P(+) cells). Treatment of JB6 P(+) cells with peonidin inhibited TPA-induced COX-2 expression, and also decreased TPA-induced neoplastic transformation and blocked TPA-induced phosphorylation of extracellular signal-regulated kinases (ERKs) in the cells. The inhibition of the signaling mechanism regulating the activation of ERKs strongly suggests that peonidin exhibits chemopreventive as well as anti-inflammatory activities.

(465) Inhibitory Effect of Lingonberry (Vaccinium vitis-idaea L.) Extracts on Activator Protein-1, Nuclear Factor-KappaB, and Mitogen-activated Protein Kinases

The effects of lingonberry (Vaccinium vitis-idaea L.) extracts on activator protein-1 (AP-1), nuclear factor-kappaB (NF-κB), and mitogen-activated protein kinases (MAPKs) were evaluated. Pretreatment of JB6 P+ mouse epidermal cells with lingonberry extracts produced a dose-dependent inhibition of AP-1 and NF-κB induced by either 12-O-tetradecanoylphorbol-13-acetate (TPA) or ultraviolet-B (UVB) light. Lingonberry extracts blocked UVB-induced phosphorylation of MAPK family members ERK1, ERK2, and p38, but not JNK. Lingonberry extracts also prevented TPA-induced phosphorylation of ERK1 and ERK2. Results of soft agar assays indicated that lingonberry extracts suppressed TPA-induced neoplastic transformation of JB6 P+ cells in a dose-dependent manner. Lingonberry extracts also induced the apoptosis of human leukemia HL-60 cells in a dose-independent manner. These results suggest that ERK1 and ERK2 may be inhibited by lingonberries, which results in suppression of AP-1 and neoplastic transformation in JB6 P+ cells and causes cancer cell death by an apoptotic mechanism in human leukemia HL-60 cells.

Antioxidant Activity in Lingonberries (Vaccinium vitis-idaea L.) and Its Inhibitory Effect on Activator Protein-1, Nuclear Factor-κB, and Mitogen-Activated Protein Kinases Activation

Lingonberry has been shown to contain high antioxidant activity. Fruits from different cultivars of lingonberry (Vaccinium vitis-idaea L.) were evaluated for fruit quality, antioxidant activity, and anthocyanin and phenolic contents. The fruit soluble solids, titratable acids, antioxidant capacity, and anthocyanin and phenolic contents varied with cultivars. Lingonberries contain potent free radical scavenging activities for DPPH*, ROO*, *OH, and O2*- radicals. Pretreatment of JB6 P+ mouse epidermal cells with lingonberry extracts produced a dose-dependent inhibition on the activation of activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB) induced by either 12-O-tetradecanoylphorbol-13-acetate (TPA) or ultraviolet-B (UVB). Lingonberry extract blocked UVB-induced phosphorylation of the mitogen-activated protein kinase (MAPK) signaling members ERK1, ERK2, p38, and MEK1/2 but not JNK. Lingonberry extract also prevented TPA-induced phosphorylation of ERK1, ERK2, and MEK1/2. Results of soft agar assays indicated that lingonberry extract suppressed TPA-induced neoplastic transformation of JB6 P(+) cells in a dose-dependent manner. Lingonberry extract also induced the apoptosis of human leukemia HL-60 cells in a dose-independent manner. These results suggest that ERK1, ERK2, and MEK1/2 may be the primary targets of lingonberry that result in suppression of AP-1, NF-kappaB, and neoplastic transformation in JB6 P(+) cells and causes cancer cell death by an apoptotic mechanism in human leukemia HL-60 cells.

Requirement for phosphatidylinositol 3-kinase in epidermal growth factor-induced AP-1 transactivation and transformation in JB6 P+ cells.

Phosphatidylinositol 3-kinase (PI 3-kinase) plays a role in a variety of biological processes, including regulation of gene expression, cell growth, and differentiation. However, little is known about its role in the cytoplasmic events involved in epidermal growth factor (EGF)-induced transduction of signals to the transcriptional machinery of the nucleus and in EGF-induced cell transformation. In this study, we examined whether PI 3-kinase is a mediator for the activation of AP-1 and neoplastic transformation by EGF in the murine epidermal cell line JB6. The results showed the following. (i) EGF not only induced a high level of PI 3-kinase activity by itself but also enhanced insulin-induced PI 3-kinase activity in JB6 P+ cells, the EGF-induced PI-3 kinase activity could be blocked by constitutive overexpression of a dominant negative P85 subunit of PI 3-kinase (deltaP85), and insulin could markedly promote EGF-induced AP-1 activity in a dose-dependent manner in JB6 P+ cells as well as promote EGF-induced JB6 P+ cell transformation. (ii) Inhibition of PI-3 kinase with wortmannin or LY294002 markedly decreased the AP-1 activity induced by insulin, EGF, or EGF and insulin in a dose-dependent manner, while wortmannin did not block UVB-induced AP-1 activity. (iii) AP-1 activation by insulin, EGF, or EGF and insulin could be completely inhibited by overexpression of deltaP85 in all the dose and time courses studied. (iv) Inhibitors of PI 3-kinase (wortmannin and LY294002) and stable overexpression of deltaP85 inhibited EGF-induced transformation but had no significant inhibitory effect on cell proliferation induced by EGF or EGF and insulin. These results demonstrate for the first time that PI 3-kinase appears to be required for EGF- or insulin-induced AP-1 transactivation and cell transformation but not cell proliferation in JB6 cells.

Blocking of tumor promoter-induced AP-1 activity inhibits induced transformation in JB6 mouse epidermal cells.

AP-1 transcriptional activity is stimulated by the transformation promoters phorbol 12-myristate 13-acetate ("12-O-tetradecanoylphorbol 13-acetate," TPA) and epidermal growth factor (EGF) in promotion-sensitive (P+) but not in promotion-resistant (P-) JB6 mouse epidermal cell lines. Although TPA stimulates expression of the jun and fos family genes, only c-jun expression shows higher elevation in P+ cells than in P- cells. The present study tests the hypothesis that induced AP-1 activity is required for tumor promoter-induced transformation in JB6 P+ cells. Both retinoic acid and the glucocorticoid fluocinolone acetonide inhibited basal and TPA-induced AP-1 activities that were tested with a stromelysin promoter-chloramphenicol acetyltransferase reporter gene in P+ cells. Since both retinoic acid and fluocinolone acetonide are active in inhibiting TPA-induced anchorage-independent transformation of P+ cells in the dose range that blocks TPA-induced AP-1 activity, their antipromoting effects may occur through inhibition of AP-1 activity. To test the hypothesis with a more specific inhibitor, stable clonal transfectants of P+ cells expressing dominant negative c-jun mutant encoding a transcriptionally inactive product were analyzed. All transfectants showed a block in TPA and EGF induction of AP-1 activity. All transfectants also showed inhibition of TPA-induced transformation, and most transfectants showed a block in EGF-induced transformation. These results indicate that AP-1 activity is required for TPA- or EGF-induced transformation. This work demonstrates that a specific block in induced AP-1 activity inhibits tumor promoter-induced transformation.