Transformation Of JB6 Cells Research Articles

Eupatilin inhibits EGF-induced JB6 cell transformation by targeting PI3K.

Phosphatidylinositol 3-kinases (PI3Ks) are lipid kinases that play fundamental roles in regulation of multiple signaling pathways, including cell proliferation, survival and cell cycle. Increasing evidence has shown that abnormal activation of PI3K pathway contributes to tumorigenesis and progression of various malignant tumors. Therefore, it is an attractive target of chemoprevention and chemotherapy. Eupatilin, a natural flavone compound extracted from Artemisia vulgaris, has antitumor and anti-inflammation efficacy. However, the direct target(s) of eupatilin in cancer chemoprevention are still elusive. In the present study, we reported eupatilin suppressed JB6 cell proliferation and its EGF-induced colony formation. Eupatilin attenuated phosphorylation of PI3K downstream signaling molecules. Downregulation ofcyclin D1 expression and arresting in G1 phase were induced through eupatilin treatment. Furthermore, we found it could bind to the p110α, a catalytic subunit of PI3K, by computational docking methods. Pull down assay outcomes also verified the binding of eupatilin with PI3K. Taken together, our results suggest that epatilin is a potential chemopreventive agent in inhibition of skin cell transformation by targeting PI3K.

Growth-suppressive effect of suberoylanilide hydroxamic acid (SAHA) on human oral cancer cells.

The histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) has been reported to exhibit anticancer activities in various cancer cell types, but as yet there are few reports on the anticancer effects of SAHA in oral squamous cell carcinoma (OSCC)-derived cells and xenograft models. The anti-proliferative and apoptotic activities of SAHA were assessed in human HSC-3 and HSC-4 (OSCC)-derived cell lines and JB6 normal mouse skin-derived epidermal cells using histone acetylation, soft agar colony formation, trypan blue exclusion, 4'-6-diamidino-2-phenylindole (DAPI) staining, Live/Dead viability/cytotoxicity and Western blot analyses. We found that SAHA treatment resulted in hyperacetylation of histones H2A and H3 and a concomitant decrease in the viability of HSC-3 and HSC-4 cells. SAHA also significantly inhibited the neoplastic transformation of JB6 cells treated with TPA, whereas the viability of these cells was not affected by this treatment. Additionally, we found that SAHA suppressed the anchorage-independent growth (colony forming capacity in soft agar) of HSC-3 and HSC-4 cells. DAPI staining, Live/Dead and Western blot analyses revealed that SAHA can induce caspase-dependent apoptosis in HSC-3 and HSC-4 cells. We also found that SAHA treatment led to inhibition of ERK phosphorylation, and that two MEK inhibitors potentiated SAHA-mediated apoptosis. Okadaic acid treatment inhibited SAHA-mediated apoptosis in both the HSC-3 and HSC-4 cell lines, wheras SAHA induced a profound in vivo inhibition of tumor growth in HSC-3 xenografts. Our results indicate that the ERK signaling pathway may constitute a critical denominator of SAHA-induced apoptosis in OSCC-derived cells and that SAHA may have therapeutic potential for OSCC.

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

Blocking of JB6 cell transformation by tanshinone IIA: epigenetic reactivation of Nrf2 antioxidative stress pathway.

Increasing numbers of natural products have been found to possess anticancer effects. Nuclear factor erythroid-2-related factor-2 (Nrf2) is a master regulator of the antioxidative stress response, and our previous studies found that epigenetic modification of the Nrf2 gene appears to be a critical mechanism. Salvia miltiorrhiza, a Chinese herbal medicine widely used in Asian countries, has been shown to possess anticancer and antioxidant effects. Tanshinone IIA (TIIA), an active component in S. miltiorrhiza, has been reported to activate Nrf2 pathway. The objective of this study was to investigate the epigenetic regulation of Nrf2 by TIIA in mouse skin epidermal JB6 cells and the functional consequences for cell transformation. TIIA was found to induce antioxidant response element-luciferase and upregulate the mRNA and protein levels of Nrf2 and Nrf2 downstream target genes HO-1 and NQO-1. TIIA decreased the colony formation of JB6 cells by approximately 80%. TIIA decreased the protein levels of DNMT1, DNMT3a, DNMT3b, and HDAC3 and inhibited the enzymatic activity of HDACs. Bisulfite genomic sequencing indicated that TIIA demethylated the first five CpGs in the promoter region of the Nrf2 gene. Chromatin immunoprecipitation assays showed that TIIA treatment increased the recruitment of RNA polymerase II at Nrf2 transcription start site but had limited effects on enrichment of Ac-H3 in Nrf2 promoter. Taken together, our results show that TIIA activates the Nrf2 signaling pathway and induces epigenetic demethylation of the CpGs of Nrf2. The epigenetic reactivation of the Nrf2 signaling pathway by TIIA could potentially contribute to the attenuation of JB6 cellular transformation and anticancer effects.

ANTIOXIDANTS AND HEALTH BENEFITS OF STRAWBERRIES

Strawberries are a good source of natural antioxidants and antioxidant enzymes which provide protection against harmful free radicals and play an important role in protecting human health. Free radicals or reactive oxygen species (ROS) are generated as by-products of normal metabolism and under stressful conditions. Increased levels of these free radicals or ROS could create oxidative stress, which leads to a variety of biochemical and physiological injuries and often results in impairment of metabolism, and eventually cell death. Successful prevention of ROS activities will be the key to controlling morbidity and mortality from chronic diseases. Therefore, eating more fruits and vegetables including strawberries has been associated with lower incidence and mortality rates of cancer, heart disease and a number of other human diseases. We have found that strawberry fruit extracts (SFE) exhibit chemopreventive and chemotherapeutic activities. They significantly inhibited activation of activator protein-1 (AP-1), nuclear factor-KappaB (NF-IoB), and mitogen-activated protein kinases (MAPKs) signaling induced by UV or 12-O-tetradecanolyphorbol-13-acetate (TPA). Furthermore, SFEs inhibited TPA-induced neoplastic transformation in JB6 cells. SFEs specifically induced apoptosis of cancerous HL-60 cells, but had no effect on normal human peripheral blood mononuclear cells. SFEs also inhibited proliferation of A549 cells, a human lung cancer cell line with little cytotoxicity to normal tissue. These results suggest that the chemopreventive effects of strawberries may be through their antioxidant properties by blocking ROS-mediated AP-1, NF- IoB, and MAPK activation. Many factors could affect antioxidant enzymes and antioxidant activity in strawberries, such as genotype variation and maturity, pre-harvest conditions, culture practices, postharvest handling, storage and processing. Optimizing these antioxidant systems in strawberries will be helpful for improving human health.

Chemopreventive and chemotherapeutic effect of a novel histone deacetylase inhibitor, by specificity protein 1 in MDA-MB-231 human breast cancer cells

Histone deacetylase inhibitors (HDACi) have been reported to have potent chemopreventive activity because of their effects on the inhibition of cell growth and apoptosis in human cancer cell lines. In the present study, we investigated the apoptotic effect of a novel HDACi, Ky2, and its molecular mechanism in MDA-MB-231 human breast cancer cells in vitro. The chemopreventive effects of Ky2 in MDA-MB-231 cells were evaluated using the MTS assay, anchorage-independent cell transformation assay, DAPI staining, western blot analysis, reverse transcriptase-PCR, and small interfering RNA. Ky2 enhanced histone acetylation and decreased cell viability. Ky2 induced apoptosis evidenced by nuclear condensation and fragmentation, the accumulation of sub-G1 phase, and caspase-dependent PARP cleavage. In addition, Ky2 released cytochrome c from mitochondria to cytosol through the regulation of mitochondria-related proteins (Bid, Bim, and Bcl-xL). Ky2 markedly decreased the level of Sp1 protein expression through both the decrease of Sp1 mRNA level and proteasome-dependent protein degradation. Interestingly, the apoptotic effect of Ky2 is more potent than SAHA, a well-known HDACi. Furthermore, the knockdown of Sp1 protein by Sp1-specific inhibitor, mithramycin A, and siRNA resulted in the alteration of truncated Bid and Bim to induce apoptosis. Furthermore, Ky2 significantly decreased TPA-induced or EGF-induced neoplastic cell transformation in JB6 cells. Our results suggest that Ky2 may be a potential chemopreventive and chemotherapeutic agent by modulating Sp1 in human breast cancer cells.

Metallic Nickel Nanoparticles May Exhibit Higher Carcinogenic Potential than Fine Particles in JB6 Cells

While numerous studies have described the pathogenic and carcinogenic effects of nickel compounds, little has been done on the biological effects of metallic nickel. Moreover, the carcinogenetic potential of metallic nickel nanoparticles is unknown. Activator protein-1 (AP-1) and nuclear factor-κB (NF-κB) have been shown to play pivotal roles in tumor initiation, promotion, and progression. Mutation of the p53 tumor suppressor gene is considered to be one of the steps leading to the neoplastic state. The present study examines effects of metallic nickel fine and nanoparticles on tumor promoter or suppressor gene expressions as well as on cell transformation in JB6 cells. Our results demonstrate that metallic nickel nanoparticles caused higher activation of AP-1 and NF-κB, and a greater decrease of p53 transcription activity than fine particles. Western blot indicates that metallic nickel nanoparticles induced a higher level of protein expressions for R-Ras, c-myc, C-Jun, p65, and p50 in a time-dependent manner. In addition, both metallic nickel nano- and fine particles increased anchorage-independent colony formation in JB6 P+ cells in the soft agar assay. These results imply that metallic nickel fine and nanoparticles are both carcinogenetic in vitro in JB6 cells. Moreover, metallic nickel nanoparticles may exhibit higher carcinogenic potential, which suggests that precautionary measures should be taken in the use of nickel nanoparticles or its compounds in nanomedicine.

Aaptamines from the marine sponge Aaptos sp. display anticancer activities in human cancer cell lines and modulate AP-1-, NF-κB-, and p53-dependent transcriptional activity in mouse JB6 Cl41 cells.

Aaptamine (8,9-dimethoxy-1H-benzo[de][1,6]naphthyridine) is a marine natural compound possessing antioxidative, antimicrobial, antifungal, and antiretroviral activity. Earlier, we have found that aaptamine and its derivatives demonstrate equal anticancer effects against the human germ cell cancer cell lines NT2 and NT2-R and cause some changes in the proteome of these cells. In order to explore further the mechanism of action of aaptamine and its derivatives, we studied the effects of aaptamine (1), demethyl(oxy)aaptamine (2), and isoaaptamine (3) on human cancer cell lines and on AP-1-, NF-κB-, and p53-dependent transcriptional activity in murine JB6 Cl41 cells. We showed that compounds 1–3 demonstrate anticancer activity in THP-1, HeLa, SNU-C4, SK-MEL-28, and MDA-MB-231 human cancer cell lines. Additionally, all compounds were found to prevent EGF-induced neoplastic transformation of murine JB6 Cl41 cells. Nuclear factors AP-1, NF-κB, and p53 are involved in the cellular response to high and nontoxic concentrations of aaptamine alkaloids 1–3. Furthermore, inhibition of EGF-induced JB6 cell transformation, which is exerted by the compounds 1–3 at low nontoxic concentrations of 0.7–2.1 μM, cannot be explained by activation of AP-1 and NF-κB.

Osteopontin mediates tumorigenic transformation of a preneoplastic murine cell line by suppressing anoikis: An Arg‐Gly‐Asp‐dependent‐focal adhesion kinase‐caspase‐8 axis

Osteopontin (OPN), an adhesive, matricellular glycoprotein, is a rate-limiting factor in tumor promotion of skin carcinogenesis. With a tumor promotion model, the JB6 Cl41.5a cell line, we have shown that suppressing 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced OPN expression markedly inhibits TPA-induced colony formation in soft agar, an assay indicative of tumorigenic transformation. Further, the addition of exogenous OPN promotes colony formation of these cells. These findings support a function of OPN in mediating TPA-induced neoplastic transformation of JB6 cells. In regard to the mechanism of action by OPN, we hypothesized that, for JB6 cells grown in soft-agar, secreted OPN induced by TPA stimulates cell proliferation and/or prevents anoikis to facilitate TPA-induced colony formation. Analyses of cell cycle and cyclin D1 expression, and direct cell counting of JB6 cells treated with OPN indicate that OPN does not stimulate cell proliferation relative to non-treated controls. Instead, at 24 h, OPN decreases anoikis by 41%, as assessed by annexin V assays. Further, in suspended cells OPN suppresses caspase-8 activation, which is mediated specifically through its RGD-cell binding motif that transduces signals through integrin receptors. Transfection studies with wild-type and mutant focal adhesion kinases (FAK) and Western blot analyses suggest that OPN suppression of caspase-8 activation is mediated through phosphorylation of FAK at Tyr(861). In summary, these studies indicate that induced OPN is a microenvironment modulator that facilitates tumorigenic transformation of JB6 cells by inhibiting anoikis through its RGD-dependent suppression of caspase-8 activity, which is mediated in part through the activation of FAK at Tyr(861).

Abstract 2234: Catechol suppresses EGF-induced cell transformation by inhibiting ERK2 activity as confirmed by a crystallographic study.

Abstract Catechol (pyrocathechol, 1,2-dihydroxybenzene) is a chemical that is used by industry in the manufacture of certain products. It also occurs naturally in many fruits such as apple, apricot, grape, bananas, pear, plum, mango, avocado, potato, and mushrooms. Browning of fruit occurs because of a chemical reaction between catechol and oxygen. Even though, catechol has toxicity at a high dose as shown by an in vivo study, the Environmental Protection Agency has not classified catechol with respect to potential carcinogenicity. Whether a lower concentration (< 40 μM) of catechol treatment has effects on carcinogenesis is not known. We found that catechol binds to ERK2 and inhibits its activity. The extracellular signal-regulated kinases (ERKs) belong to the mitogen-activated protein (MAP) kinase family, and regulate important biological processes such as cell growth, proliferation, differentiation, and transcription regulation. ERK2 is activated in response of UV, cytokines and growth factors, such as epidermal growth factor (EGF) and insulin-like growth factor. Constitutive activation of ERK2 is observed in many human cancers such as colon, breast, lung and skin cancers. In this study we present the co-crystal structure of ERK2 bound with catechol in the ATP-active site. The conserved amino acid residues within the hinge region that are involved in the interaction of ERK and its inhibitors make the contacts with catechol. Contacts include side chain of Q105, and the main chains of the D106 and M108 residues. We found that catechol inhibits ERK2 in vitro kinase activity more than two-fold at a 20 μM concentration. This was confirmed by the observed decrease of the phosphorylation of Elk1, a specific downstream target of ERK2. In addition, 20 μM catechol inhibited EGF-induced JB6 cell transformation. Interestingly, JB6 cell proliferation was not affected at 20 μM concentration. Taken together, the results showed that catechol inhibits EGF-induced cell transformation without any toxicity through the inhibition of ERK2 activity. Citation Format: Do Young Lim, Margarita Malakhova, Igor Kurinov, Seung Ho Shin, Mee-Hyun Lee, Ann M. Bode, Zigang Dong. Catechol suppresses EGF-induced cell transformation by inhibiting ERK2 activity as confirmed by a crystallographic study. [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 2234. doi:10.1158/1538-7445.AM2013-2234

Mithramycin A Inhibits Myeloid Cell Leukemia-1 to Induce Apoptosis in Oral Squamous Cell Carcinomas and Tumor Xenograft through Activation of Bax and Oligomerization

In several human malignancies, overexpression of myeloid cell leukemia-1 (Mcl-1) confers resistance to induction of apoptosis; however, Mcl-1-mediated inhibition of apoptosis in oral squamous cell carcinoma (OSCC) is not fully understood and has been investigated in this study. The Mcl-1 promoter activators (TPA) and epidermal growth factor (EGF) enhanced neoplastic transformation of JB6 cells and this response was accompanied by enhanced expression of Mcl-1, and knockdown of Mcl-1 by RNA interference (RNAi) decreased JB6 cell transformation. In the same cell line, we also demonstrated that mithramycin A (Mith) decreased TPA-induced JB6 cell transformation and Mcl-1 expression. Mcl-1 was overexpressed in human oral tumors compared with normal oral mucosa and also in several OSCC cell lines including HN22 and HSC-4 cells. Treatment of these cells with Mith also decreased Mcl-1 expression and neoplastic cell transformation, and this was accompanied by induction of several markers of apoptosis. Knockdown of Mcl-1 by RNAi also induced apoptotic cell death. The downregulation of Mcl-1 by Mith and RNAi increased pro-apoptotic protein Bax, resulting in the Bax translocation into mitochondria and its oligomerization. Mith also suppressed tumor growth in vivo and induced apoptosis in tumor by also regulating expression of Mcl-1 and Bax proteins. These indicate a critical role for Mcl-1 in the growth and survival of OSCC and demonstrate that Mith may be a potential anticancer drug candidate for clinical treatment of OSCC.

Sesquiterpene lactones isolated from indigenous Middle Eastern plants inhibit tumor promoter-induced transformation of JB6 cells

BackgroundSesquiterpene lactones (SL) are plant secondary metabolites that are known for their anti-fungal, anti-bacterial, anti-inflammatory, and anti-tumor properties. Considering that several SL-derived drugs are currently in cancer clinical trials, we have tested two SL molecules, 3-β-methoxy-iso-seco-tanapartholide (β-tan) isolated from Achillea falcata and salograviolide A (Sal A) isolated from Centaurea ainetensis, for their anti-tumor properties. We used the mouse epidermal JB6P + cells as a model for tumor promotion and cellular transformation. Key players that are involved in cellular transformation and tumorigenesis are the AP-1 and NF-κB transcription factors; therefore, we assessed how β-tan and Sal A modulate their signaling pathways in JB6P + cells.MethodsThe effects of β-tan and Sal A on the growth of normal and neoplastic keratinocytes and on the tumor promotion-responsive JB6P + cells were determined using the MTT assay. Anchorage-independent cell growth transformation assays were used to evaluate the anti-tumor promoting properties of these SL molecules in JB6P + cells and dual luciferase reporter assays and western blot analysis were used to investigate their effects on tumor promoter-induced AP-1 and NF-κB activities and protein levels of key AP-1 and NF-кB target genes.Resultsβ-tan and Sal A selectively inhibited tumor promoter-induced cell growth and transformation of JB6P + cells at concentrations that do not affect JB6P + and primary keratinocytes basal cell growth. In addition, both molecules reduced basal and tumor promoter-induced NF-κB transcriptional activities, differentially regulated basal and tumor promoter-induced AP-1 transcriptional activities, and modulated key players of the AP-1 and NF-κB signaling pathways.ConclusionsThese results highlight the anti-tumor promoting properties of β-tan and Sal A. These SL molecules isolated from two plant species native to the Middle East may provide opportunities for complementary medicine practices.

C11 cyclopentenone from the ascidian Diplosoma sp. prevents epidermal growth factor-induced transformation of JB6 cells

C<sub>11</sub> cyclopentenone, 5-hydroxy-7-prop-2-en- (E)-ylidene-7,7a-dihydro-2H-cyclopenta[b] pyran-6-one, isolated earlier from the sponges and ascidians, is known as a natural product possessing antimicrobial and cytotoxic properties. However, its cancer preventive activity has not been studied. Cancer preventive and proapoptotic properties of the compound as well as its effect on the main Mitogen- Activated Protein-Kinase (MAPK) signaling pathways were examined by the methods of epidermal growth factor- induced (EGFinduced) JB6 Cl41 P+ cell transformation in soft agar, flow cytometry, and MTS test of cell viability. Results: the compound inhibits EGFinduced neoplastic JB6 Cl41 P+ cell transformation in soft agar and induces apoptosis of HL-60 and THP-1 human leukemia cells. Jun N-terminal Kinase and p38 MAPK signaling pathways are involved in the cellular response to the treatment by the compound. Conclusions: 5-hydroxy-7-prop-2-en-(E)-ylidene- 7,7a-dihydro-2H-cyclopenta[b]pyran-6- one and other related marine C11 cyclopentenones have potential for development of a new antitumor agent in cancer prophylactics and should be further investigated.

Zerumbone Induces Heme Oxygenase-1 Expression in Mouse Skin and Cultured Murine Epidermal Cells through Activation of Nrf2

Zerumbone, a sesquiterpene derived from tropical ginger, contains an electrophilic α,β-unsaturated carbonyl moiety and was found to suppress chemically induced papilloma formation in mouse skin. Here, we report that topical application of zerumbone onto dorsal skin of hairless mice induces activation of NF-E2-related factor 2 (Nrf2) and expression of heme oxygenase-1 (HO-1). We compared the levels of HO-1 protein in the skin of zerumbone-treated Nrf2 wild-type and Nrf2 knockout mice, and nrf2-deficient mice expressed HO-1 protein to a much lesser extent than the wild-type animals following topical application of zerumbone. Treatment of mouse epidermal JB6 cells with zerumbone caused a marked increase of Nrf2 nuclear translocation followed by the promoter activity of HO-1, and also enhanced direct binding of Nrf2 to the antioxidant response element. Moreover, knockdown of Nrf2 in JB6 cells diminished the zerumbone-induced upregulation of HO-1. Notably, α-humulene and 8-hydroxy-α-humulene, the structural analogues of zerumbone that lack the α,β-unsaturated carbonyl group, failed to activate Nrf2 and were unable to increase HO-1 expression. Unlike zerumbone, these nonelectrophilic analogues could not suppress the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced JB6 cell transformation and the intracellular accumulation of reactive oxygen species (ROS). Interestingly, when JB6 cells were treated with carbon monoxide-releasing molecule that mimics the HO-1 activity, the TPA-induced ROS production was markedly reduced. Taken together, these findings suggest that upregulation of HO-1 expression by zerumbone is mediated through activation of Nrf2 signaling, which provides a mechanistic basis for the chemopreventive effects of this sesquiterpene on mouse skin carcinogenesis.

Abstract 2735: Osteopontin mediates tumorigenic transformation of a murine promotable cell line through promoting cell survival by an RGD-dependent suppression of caspase 8 activity

Abstract Osteopontin (OPN) is a cell adhesive, matricellular protein shown to be an important rate-limiting factor in tumorigenesis in vivo. Further, we have previously shown that addition of exogenous OPN promoted colony formation in an anchorage-independent growth assay of JB6 Cl41.5a cells, a preneoplastic mouse promotable cell line. In addition, down-regulation of the TPA (12-0-tetradecanoylphorbol-13-acetate)-induced OPN expression markedly suppressed TPA-induced colony formation of these cells. Collectively, these findings support the role of osteopontin in mediating neoplastic transformation of JB6 cells in vitro. In the present study, we hypothesize that induced-OPN stimulates the proliferation and/or prevents anoikis of JB6 cells grown in soft-agar and consequently, facilitates colony formation. To determine whether OPN promotes cell proliferation, cell cycle analyses by flow cytometry and direct cell counting of JB6 cells treated with soluble OPN were performed. Data from both of these analyses indicated that OPN-treated cells did not promote cell proliferation compared to non-treated controls. To determine whether OPN prevents anoikis of JB6 cells, annexin V and caspases activities were assesses on cells incubated with or without OPN and suspended on agar. Annexin V analyses showed that OPN significantly decreased anoikis of JB6 cells at 24 h by 41%. In the absence of OPN, suspended JB6 cells underwent anoikis through a sequential activation of caspases 8, 9 and 3 by 1, 3 and 18 h, respectively. Also, the addition of soluble OPN to suspended JB6 cells significantly suppressed caspase 8 activation when compared to non-treated cells. We next determined whether OPN's ability to suppress caspase 8 activity was mediated through its RGD- and/or DRVLFRI-binding motifs as they are known to transduce signals through integrin and CD44 receptors, respectively. Incubation of soluble RGD peptides with suspended JB6 cells suppressed caspase 8 activity. Similar results were obtained when suspended JB6 cells were incubated with OPN only or with RGD peptides and OPN. In contrast, RGE (non-specific peptides) and DRVLFRI peptides alone did not suppress caspase 8 activity of suspended JB6 cells unless OPN was added to these peptides. These findings suggest that OPN suppression of caspase 8 activity of suspended JB6 cells is mediated through its RGD cell-binding motif interacting with integrin receptors. In conclusion, osteopontin promotes tumorigenic transformation of JB6 cells by enhancing the survival of these cells through its RGD-dependent suppression of caspase 8 activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2735. doi:10.1158/1538-7445.AM2011-2735

Activation and up‐regulation of translation initiation factor 4B contribute to arsenic‐induced transformation

Arsenic is a known human carcinogen. However, the mechanism of how arsenic induces cell transformation remains unclear. In this study, we demonstrated that long-term exposure to sodium arsenite at low-dose (2 µM) increases cell proliferation and neoplastic transformation in a mouse epidermal cell model, JB6 promotion-susceptible cells. The phosphorylation of AKT and its downstream targets, 70-kDa ribosomal protein S6 kinase (p70S6K) and translation initiation factor 4B (eIF4B), are increased in the arsenite treated cells, indicating that long-term arsenite treatment activates AKT-p70S6K signaling pathway. In addition, long-term exposure to arsenite up-regulates eIF4B expression and increases the rate of translation. Knockdown of eIF4B expression resulted in inhibition of arsenic-induced cell proliferation, transformation, and translation. Moreover, the expression of c-Myc which is up-regulated by long-term arsenite treatment is inhibited by eIF4B knockdown. Taken together, these results indicate that activation and up-regulation of eIF4B contributes to arsenic-induced transformation in JB6 cells.

Mitochondrial uncoupling inhibits p53 mitochondrial translocation in TPA-challenged skin epidermal JB6 cells.

The tumor suppressor p53 is known to be able to trigger apoptosis in response to DNA damage, oncogene activation, and certain chemotherapeutic drugs. In addition to its transcriptional activation, a fraction of p53 translocates to mitochondria at the very early stage of apoptosis, which eventually contributes to the loss of mitochondrial membrane potential, generation of reactive oxygen species (ROS), cytochrome c release, and caspase activation. However, the mitochondrial events that affect p53 translocation are still unclear. Since mitochondrial uncoupling has been suggested to contribute to cancer development, herein, we studied whether p53 mitochondrial translocation and subsequent apoptosis were affected by mitochondrial uncoupling using chemical protonophores, and further verified the results using a siRNA approach in murine skin epidermal JB6 cells. Our results showed that mitochondrial uncoupling blocked p53 mitochondrial translocation induced by 12-O-tetradecanoylphorbol 13-acetate (TPA), a known tumor promoter to induce p53-mediated apoptosis in skin carcinogenesis. This blocking effect, in turn, led to preservation of mitochondrial functions, and eventually suppression of caspase activity and apoptosis. Moreover, uncoupling protein 2 (UCP2), a potential suppressor of ROS in mitochondria, is important for TPA-induced cell transformation in JB6 cells. UCP2 knock down cells showed enhanced p53 mitochondrial translocation, and were less prone to form colonies in soft agar after TPA treatment. Altogether, our data suggest that mitochondrial uncoupling may serve as an important regulator of p53 mitochondrial translocation and p53-mediated apoptosis during early tumor promotion. Therefore, targeting mitochondrial uncoupling may be considered as a novel treatment strategy for cancer.

Abstract 5665: Zerumbone inhibits phorbol ester-induced intracellular ROS accumulation and neoplastic transformation by inducing Nrf2-driven heme oxygenase-1 expression in mouse epidermal cells

Abstract Zerumbone, derived from tropical ginger (Zingibers zerumbet, Zingiberaceae), was found to suppress chemically-induced skin tumor formation in mice. However, the molecular mechanisms underlying the chemopreventive effects of zerumbone on mouse skin carcinogenesis are poorly understood. Here, we report that topical application of zerumbone onto dorsal skin of HR-1 hairless mice induced expression of heme oxygenase-1 (HO-1) through activation of Nrf2, a major transcription factor that plays a pivotal role in regulating expression of several antioxidant/detoxifying enzymes. To verify the possible involvement of Nrf2 in zerumbone-derived induction of HO-1, we compared HO-1 protein levels in the zerumbone-treated skin of Nrf2 wild-type (+/+) and Nrf2 knock out (−/−) mice. Nrf2-deficient mice expressed HO-1 in the skin to a much lesser extent than did the wild-type animals following topical application of zerumbone (10 μmol). Moreover, zerumbone (10 μM) inhibited anchorage-independent growth as well as ROS production in mouse epidermal JB6 cells stimulated with 12-0-tetradecanoylphorbol-13-acetate (TPA; 10 ng/ml). Likewise, treatment of JB6 cells with zerumbone resulted in a markedly increased nuclear translocation of Nrf2 as well as the promoter activity of HO-1. In addition, siRNA knock down of nrf2 in JB6 cells diminished the zerumbone-induced expression of HO-1. Using a chromatin immunoprecipitation (ChIP) assay, we confirmed that zerumbone induced the direct binding of Nrf2 to antioxidant response element (ARE). We hypothesized that the electrophilic α,β-unsaturated carbonyl moiety present in zerumbone could modify critical cysteine thiols of Keap1, a cytosolic repressor of Nrf2, thereby facilitating its dissociation from Keap1 for nuclear translocation. α-Humulene and 8-hydroxy-α-humulene, structural derivatives of zerumbone that lack the α,β-unsaturated carbonyl group, were unable to activate Nrf2 and induce HO-1 expression in both mouse skin in vivo and cultured mouse epidermal cells. Unlike zerumbone, these non-electrophilic analogs failed to suppress TPA-induced JB6 cell transformation and ROS accumulation. Interestingly, treatment of JB6 cells with 50 μM carbon monoxide-releasing molecule (CORM) markedly reduced ROS accumulation indicating that carbon monoxide formed as a consequence of zerumbone-induced HO-1 expression has an important role in abolishing TPA-induced ROS generation. Taken together, the above findings suggest that up-regulation of HO-1 by zerumbone is mediated via the Nrf2 signaling pathway, which provides a mechanistic basis for the anti-tumor promoting effects of this sesquiterpene on mouse skin carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5665.

Fyn is a novel target of (−)‐epigallocatechin gallate in the inhibition of JB6 Cl41 cell transformation

The cancer preventive action of (-)-epigallocatechin gallate (EGCG), found in green tea, is strongly supported by epidemiology and laboratory research data. However, the mechanism by which EGCG inhibits carcinogenesis and cell transformation is not clear. In this study, we report that EGCG suppressed epidermal growth factor (EGF)-induced cell transformation in JB6 cells. We also found that EGCG inhibited EGF-induced Fyn kinase activity and phosphorylation in vitro and in vivo. Fyn was implicated in the process because EGF-induced JB6 cell transformation was inhibited by small interfering RNA (siRNA)-Fyn-JB6 cells. With an in vitro protein-binding assay, we found that EGCG directly bound with the GST-Fyn-SH2 domain but not the GST-Fyn-SH3 domain. The K(d) value for EGCG binding to the Fyn SH2 domain was 0.367 +/- 0.122 microM and B(max) was 1.35 +/- 0.128 nmol/mg. Compared with control JB6 Cl41 cells, EGF-induced phosphorylation of p38 MAP kinase (p38 MAPK) (Thr180/Tyr182), ATF-2 (Thr71) and signal transducer and activator of transcription 1 (STAT1) (Thr727) was decreased in siRNA-Fyn-JB6 cells. EGCG could inhibit the phosphorylation of p38 MAPK, ATF-2, and STAT1. The DNA binding ability of AP-1, STAT1, and ATF-2 was also decreased in siRNA-Fyn-JB6 cells. Overall, these results demonstrated that EGCG interacted with Fyn and inhibited Fyn kinase activity and thereby regulated EGF-induced cell transformation. Inhibition of Fyn kinase activity is a novel and important mechanism that may be involved in EGCG-induced inhibition of cell transformation.

Redox effector factor-1, combined with reactive oxygen species, plays an important role in the transformation of JB6 cells

Apurinic/apyrimidinic endonuclease/redox effector factor-1 (APE/Ref-1) is a multifunctional protein involved both in DNA base excision repair and redox regulation. Studies have suggested that abnormal Ref-1 levels and/or activities are associated with tumor progression and sensitivities to treatment, but no direct evidence has yet been published regarding the role of Ref-1 in malignant transformation. We utilized the well-documented tumor promotor-sensitive JB6 mouse epithelial cell model as well as new transformants [by ultraviolet light B (UVB), H2O2 or Cd] to study this phenomenon. Significant increases of reactive oxygen species (ROS) were observed in JB6P+ and all the transformants compared with promotor-resistant JB6P- cells. These increases were paralleled by a sustained elevation of Ref-1 expression. Further analysis exhibited a strong inverse correlation between oxidative DNA lesions [8-oxodeoxyguanosine (8-oxo-dG)] and Ref-1 levels in all JB6 cells. Notably, apoptosis occurred after knock-down of Ref-1 by small interfering RNA (siRNA)] demonstrated by a approximately 2-fold increase of Annexin V-positive JB6P+ cells. Ref-1 depletion also inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced anchorage-independent growth of JB6P+ by 40% and reduced the colony numbers of JB6P+/H2O2 and JB6P+/Cd cells. Mechanistic studies revealed that Ref-1 reduction was associated with an increase of intracellular ROS levels and a marked decrease of activator protein-1 (AP-1) transcription activities in JB6P+/H2O2 cells. This is the first report of the novel role of Ref-1 in cellular transformation. Based on the data presented here, we propose that induction of Ref-1, serving as an adaptive response to elevated ROS, plays a critical role in transformation and protects cells from excess ROS stresses through both DNA repair and activation of transcription factors such as activator protein-1.