Non-tumorigenic Human Keratinocyte Research Articles

Mechanical profile of human keratinocytes expressing HPV-18 oncogenes

During tumorigenesis, the mechanical properties of cancer cells change markedly, with decreased stiffness often accompanying a more invasive phenotype. Less is known about the changes in mechanical parameters at intermediate stages in the process of malignant transformation. We have recently developed a pre-tumoral cell model by stably transducing the immortalized but non-tumorigenic human keratinocyte cell line HaCaT with the E5, E6 and E7 oncogenes from HPV-18, one of the leading causes of cervical cancer and other types of cancer worldwide. We have used atomic force microscopy (AFM) to measure cell stiffness and to obtain mechanical maps of parental HaCaT and HaCaT E5/E6/E7-18 cell lines. We observed a significant decrease in Young's modulus in HaCaT E5/E6/E7-18 cells measured by nanoindentation in the central region, as well as decreased cell rigidity in regions of cell-cell contact measured by Peakforce Quantitative Nanomechanical Mapping (PF-QNM). As a morphological correlate, HaCaT E5/E6/E7-18 cells displayed a significantly rounder cell shape than parental HaCaT cells. Our results therefore show that decreased stiffness with concomitant perturbations in cell shape are early mechanical and morphological changes during the process of malignant transformation.

Generation of a poly-functionalized indolizine scaffold and its anticancer activity in pancreatic cancer cells

A highly efficient domino [4 + 2] annulation process was employed to construct a novel indolizine chemical scaffold. Biological investigation led us to identify 6w as a potent anticancer agent. 6w significantly inhibited cell viability in BxPC3 pancreatic cancer, MCF7 breast cancer, and PC3 prostate cancer cell lines with IC50 values of 0.47 ± 0.04, 1.82 ± 0.08 and 2.68 ± 0.08 µM, respectively. Remarkably, 6w showed a weak effect on cell viability of nontumorigenic human keratinocyte cell line HaCaT compared to the above three types of cancer cells. 6w most potently inhibited cell viability of BxPC3 cells, and 6w also potently reduced cell migration and induced apoptosis in BxPC3 cells through activation of caspase-3 and cleavage of PARP in a dose-dependent manner. These results suggest that 6w can be used for the development of potential anticancer drugs for the treatment of pancreatic cancer.

Methoxychalcones: Effect of Methoxyl Group on the Antifungal, Antibacterial and Antiproliferative Activities.

Chalcones substituted by methoxyl groups have presented a broad spectrum of bioactivities, including antifungal, antibacterial and antiproliferative effects. However, a clear and unambiguous investigation about the relevance of this substituent on the chalcone framework has not been described. The purpose of this work is to assess the antibacterial, antifungal and antiproliferative activities of the two series of seventeen synthesized regioisomeric methoxychalcones. Series I and II were constituted by chalcones substituted by methoxyl groups on rings A (5-12) and B (13-21), respectively. In addition, the library of methoxychalcones was submitted to in silico drug-likeness and pharmacokinetics properties predictions. Methoxychalcones were synthesized and their structures were confirmed by NMR spectral data analyses. Evaluations of antimicrobial activity were performed against five species of Candida, two Gram-negative and five Gram-positive species. For antiproliferative activity, methoxychalcones were evaluated against four human tumorigenic cell lines, as well as human non-tumorigenic keratinocytes. Drug-likeness and pharmacokinetics properties were predicted using Molinspiration and PreADMET toolkits. In general, chalcones of series I are the most potent antifungal, antibacterial and antiproliferative agents. 3', 4', 5'-Trimethoxychalcone (12) demonstrated potent antifungal activity against Candida krusei (MIC = 3.9 μg/mL), eight times more potent than fluconazole (reference antifungal drug). 3'-Methoxychalcone (6) displayed anti-Pseudomonas activity (MIC = 7.8 μg/mL). 2',5'-Dimethoxychalcone (9) displayed potent antiproliferative effect against C-33A (cervix), A-431 (skin) and MCF-7 (breast), with IC50 values ranging from 7.7 to 9.2 μM. Its potency was superior to curcumin (reference antiproliferative compound), which exhibited IC50 values ranging from 10.4 to 19.0 μM. Our studies corroborated the relevance of methoxychalcones as antifungal, antibacterial and antiproliferative agents. In addition, we elucidated influence of the position and number of methoxyl groups toward bioactivity. In silico predictions indicated good drug-likeness and pharmacokinetics properties to the library of methoxychalcones.

A natural occurring bifunctional CPD/(6-4)-photolyase from the Antarctic bacterium Sphingomonas sp. UV9.

Photolyases are flavoproteins that repair ultraviolet-induced DNA lesions (cyclobutane pyrimidine dimer or CPD, and pyrimidine (6-4) pyrimidone photoproducts or (6-4)-PPs), using blue light as an energy source. These enzymes are substrate specific, meaning that a specific photolyase repairs either a CPD or a (6-4)-PP. In this work, we produced a class II CPD-photolyase (called as PhrSph98) from the Antarctic bacterium Sphingomonas sp. UV9 by recombinant DNA technology and we purified the enzyme using immobilized metal affinity chromatography. By using an immunochemistry assay, with monoclonal antibodies against CPD and (6-4)-PP, we found that PhrSph98 repairs both DNA lesions. The result was confirmed by immunocytochemistry using immortalized non-tumorigenic human keratinocytes. Results from structure prediction, pocket computation, and molecular docking analyses showed that PhrSph98 has the two expected protein domains (light-harvesting antenna and a catalytic domain), a larger catalytic site as compared with photolyases produced by mesophilic organisms, and that both substrates fit the catalytic domain. The results obtained from predicted homology modeling suggest that the electron transfer pathway may occur following this pathway: Y389-W369-W390-F376-W381/FAD. The evolutionary reconstruction of PhrSph98 suggests that this is a missing link that reflects the transition of (6-4)-PP repair into the CPD repair ability for the class II CPD-photolyases. To the best of our knowledge, this is the first report of a naturally occurring bifunctional, CPD and (6-4)-PP, repairing enzyme. KEY POINTS: • We report the first described bifunctional CPD/(6-4)-photoproducts repairing enzyme. The bifunctional enzyme reaches the nuclei of keratinocyte and repairs the UV-induced DNA damage. The enzyme should be a missing link from an evolutionary point of view. The enzyme may have potential uses in the pharmaceutical and cosmetic industries.

Silver Nanoparticles Synthesized Using Carica papaya Leaf Extract (AgNPs-PLE) Causes Cell Cycle Arrest and Apoptosis in Human Prostate (DU145) Cancer Cells.

Treatment of cancer has been limited by the poor efficacy and toxicity profiles of available drugs. There is a growing demand to develop alternative approaches to combat cancer such as use of nano-formulation-based drugs. Here, we report biosynthesis and characterization of silver nanoparticles (AgNPs) with papaya leaf extract (PLE) and its anti-cancer properties against different human cancer cells. Purified nanoparticles were characterized by standard techniques, such as TEM, STM, SEM, EDS, XRD, and FTIR. Furthermore, cytotoxic activity of AgNPs-PLE was carried out against different human cancer cells and non-tumorigenic human keratinocytes cells. AgNPs-PLE when compared with AgNPs-citric acid or PLE showed better efficacy against cancer cells and was also relatively less toxic to normal cells. Treatment of DU145 cells with AgNPs-PLE (0.5-5.0μg/ml) for 24-48h lowered total cell number by 24-36% (P < 0.05). Inhibition of cell growth was linked with arrest of cell cycle at G2/M phase at 24h, while G1 and G2/M phase arrests at 48h. ROS production was observed at earlier time points in presence of AgNPs-PLE, suggesting its role behind apoptosis in DU145 cells. Induction of apoptosis (57%) was revealed by AO/EB staining in DU145 cells along with induction of Bax, cleaved caspase-3, and cleaved PARP proteins. G1-S phase cell cycle check point marker, cyclin D1 was down-regulated along with an increase in cip1/p21 and kip1/p27 tumor suppressor proteins by AgNPs-PLE. These findings suggest the anti-cancer properties of AgNPs-PLE.

Investigation of Antioxidant and In Vitro Wound Healing Activity of Fulvic Acid

This study aimed to investigate the antioxidant and in vitro wound healing activities of fulvic acid (FA) purified from Hüsamlar leonardite in Muğla/Turkey. Purified FA was analyzed with FT/IR spectrophotometer and flame photometer for determining functional groups and cation impurities, respectively. 1, 5, 10, 20, 30, 50, 100 μg mL-1 concentrations of FA were tested for antioxidant activity (by using DPPH radical scavenging and H2O2 scavenging assays). Also, effect of FA on BJ human foreskin fibroblast and HaCaT spontaneously immortalized non-tumorigenic human keratinocyte cells proliferation was tested by in vitro MTT and WST-8 assays and on cell migration by wound healing assay (scratch assay). The results of the study show that the FA has a low DPPH radical scavenging activity, but it exhibited high H2O2 scavenging activity at low concentrations. The effect of FA on the proliferation of BJ and HaCaT cells varied according to the cell type, FA concentration and treatment time. However, it accelerated wound healing by increasing cell migration, especially in HaCaT cells. 1 and 10 μg mL-1 FA had a significant wound healing effect on BJ cells, and all concentrations of FA had a significant wound healing effect on HaCaT cells at the end of the 24 h treatments.

158 Triggering immortalized keratinocyte growth arrest by protein kinase C delta activation

Normal epidermal keratinocyte (KC) differentiation is marked by irreversible cell cycle exit and a step-wise transition through the suprabasal layers of the epidermis. In contrast, KCs in both actinic keratosis and squamous cell carcinomas (SCCs) continue to proliferate in the suprabasal compartment, indicating a defect in their growth arrest signaling. Activation of multiple protein kinase C (PKC) isoforms have been associated with KC cell cycle withdrawal. To study the involvement of PKC in KC growth arrest, we evaluated the effects of the PKC agonist 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on normal human epidermal keratinocytes (NHEKs) and HaCaT cells, an immortalized, non-tumorigenic human keratinocyte cell line. Treating NHEKs with 10 nM TPA for as little as 15 minutes induced complete growth arrest over a 7 day period while untreated KCs underwent a 93-fold increase in cell number (p<0.005). 10 nM TPA treatment of HaCaT cells did not induce any growth arrest, indicating that immortalized KCs have defective PKC-dependent growth arrest signaling. To test if PKCδ activation was able to enforce growth arrest in HaCaT cells, we transduced HaCaT cells with retroviruses encoding either EGFP or a PKCδ-EGFP fusion and sorted EGFP positive cells by flow cytometry. HaCaT-EGFP cells behaved similar to HaCaTs and were not growth arrested by TPA treatment. In contrast, HaCaT-PKCδ-EGFP treated with TPA increased in cell number only 3-fold compared to a 15-fold increase in untreated HaCaT-PKCδ-EGFP over a 4 day growth assay. Activation of PKCδ in HaCaT-PKCδ-EGFP cells was confirmed by observing TPA-induced translocation of PKCδ-EGFP to the plasma membrane and increased S299 autophosphorylation. TPA-induced growth inhibition in HaCaT-PKCδ-EGFP cells was due to arrest in G1 and G2/M, not apoptosis. Global phosphoproteomics are being employed to identify PKCδ signaling pathways that can overcome the defective growth arrest in immortalized keratinocytes and may have therapeutic benefit for the treatment of actinic keratosis and SCCs.

Abstract 100: Interleukin 1 receptor type II upregulates MMP1 expression and increases motility of keratinocytes cells

Abstract We have shown that long-term exposure to low doses of inorganic arsenite (iAs) induced malignant transformation of non-tumorigenic human keratinocytes, HaCaT cells. We further demonstrated that enhanced expression of a decoy type II receptor of interleukin-1 (IL1R2) in transformed keratinocytes mediated through IL-6 and VEGFR to increase the migration and invasion activities, indicating the oncogenic potential of IL1R2. In this present study, we further explored how IL1R2 enhances the cell motility in human keratinocytes. As described earlier, long-term and low dose iAs (0, 0.5 and 1 μM) exposed HaCaT cells were designated as HaCaT A0, A1 and A2 cells, respectively. The tumorigenic cell line, T4R2, was derived from the tumor in mice implanted with HaCaT A2 cells. By growing HaCaT A0, A1, A2 and T4R2 cells on an organotypic culture system using basal medium, we found that T4R2 cells formed multilayered morphology. By aid of siRNA and ectopic expression techniques, we further demonstrated that increased expression of IL1R2 in T4R2 cells was associated with increased matrix metalloproteinase-1 (MMP1) and enhanced cell motility. The roles of MMP1 on supporting the organotypic growth of T4R2 cells were confirmed by using the stably MMP1-silenced T4R2 cells (T4R2-shMMP1). The cell motility of T4R2-shMMP1 cells was also significantly reduced. Furthermore, IL1R2-induced MMP1 upregulation was remarkably inhibited by inhibitors of MAPK/ERK signaling. Our present results revealed that MMP1 was one of downstream targets of IL1R2 which plays a crucial role on the progression of malignancy. Therefore, IL1R2 may be a target for cancer intervention. Citation Format: Hsiu-Hua Wang, Te-Chang Lee. Interleukin 1 receptor type II upregulates MMP1 expression and increases motility of keratinocytes 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 100.

Alpha-Tocopheryl Succinate Selectively Affects the Morphology and Motility of Normal and Tumor Epithelial Cells

Alpha-tocopheryl succinate (α-TS) is a vitamin-E derivative with potential antitumor properties. In this study, we have investigated the effect of α-TS on the morphology and motility of the A431 human epidermoid carcinoma cell line and HaCaT immortalized nontumorigenic human keratinocytes. Phase contrast and fluorescent microscopy, in vitro wound healing assay (scratch test), and scanning electron microscopy have been used. In both cell lines, alteration of cell shape, actin-cytoskeleton reorganization, cell-surface smoothing, disappearance of microvillae, and cell-motility inhibition have been shown. These effects are similar in both cell lines, but in the HaCaT cell line they occur after treatment with higher α-TS doses than for A431cells. Thus, we have demonstrated the selectivity of the effect of α-TS on tumor cells derived from human-skin stratified epithelia. The results of this work may be useful for further investigations focused on development of antitumor agents with selective action toward tumor cells.

Piperlongumine and p53-reactivator APR-246 selectively induce cell death in HNSCC by targeting GSTP1.

TP53 mutations frequently occur in head and neck squamous cell carcinoma (HNSCC) patients without human papillomavirus infection. The recurrence rate for these patients is distinctly high. It has been actively explored to identify agents that target TP53 mutations and restore wild-type (WT) TP53 activities in HNSCC. PRIMA-1 (p53-Reactivation and Induction of Massive Apoptosis-1) and its methylated analogue PRIMA-1Met (also called APR-246) were found to be able to reestablish the DNA-binding activity of p53 mutants and reinstate the functions of WT p53. Herein we report that piperlongumine (PL), an alkaloid isolated from Piper longum L., synergizes with APR-246 to selectively induce apoptosis and autophagic cell death in HNSCC cells, whereas primary and immortalized mouse embryonic fibroblasts (MEFs) and spontaneously immortalized non-tumorigenic human skin keratinocytes (HaCat) are spared from the damage by the cotreatment. Interestingly, PL-sensitized HNSCC cells to APR-246 are TP53 mutation-independent. Instead, we demonstrated that glutathione S-transferase pi 1 (GSTP1), a GST family member that catalyzes the conjugation of GSH with electrophilic compounds to fulfill its detoxification function, is highly expressed in HNSCC tissues. Administration of PL and APR-246 significantly suppresses GSTP1 activity, resulting in the accumulation of ROS, depletion of GSH, elevation of GSSG, and DNA damage. Ectopic expression of GSTP1 or pretreatment with antioxidant N-acetyl-L-cysteine (NAC) abrogates the ROS elevation and decreases DNA damage, apoptosis, and autophagic cell death prompted by PL/APR-246. In addition, administration of PL and APR-246 impedes UMSCC10A xenograft tumor growth in SCID mice. Taken together, our data suggest that HNSCC cells are selectively sensitive to the combination of PL and APR-246 due to a remarkably synergistic effect of the cotreatment in the induction of ROS by suppression of GSTP1.

Biochemical and functional characterization of the human tissue kallikrein 9.

Human tissue kallikrein 9 (KLK9) is a member of the kallikrein-related family of proteases. Despite its known expression profile, much less is known about the functional roles of this protease and its implications in normal physiology and disease. We present here the first data on the biochemical characterization of KLK9, investigate parameters that affect its enzymatic activity (such as inhibitors) and provide preliminary insights into its putative substrates. We show that mature KLK9 is a glycosylated chymotrypsin-like enzyme with strong preference for tyrosine over phenylalanine at the P1 cleavage position. The enzyme activity is enhanced by Mg2+ and Ca2+, but is reversibly attenuated by Zn2+ KLK9 is inhibited in vitro by many naturally occurring or synthetic protease inhibitors. Using a combination of degradomic and substrate specificity assays, we identified candidate KLK9 substrates in two different epithelial cell lines [the non-tumorigenic human keratinocyte cells (HaCaT) and the tumorigenic tongue squamous carcinoma cells (SCC9)]. Two potential KLK9 substrates [KLK10 and midkine (MDK)] were subjected to further validation. Taken together, our data delineate some functional and biochemical properties of KLK9 for future elucidation of the role of this enzyme in health and disease.

Metabolite Profile and Antiproliferative Effects in HaCaT Cells of a Salix reticulata Extract.

Phenolic constituents of Salix reticulata (Salicaceae) and antiproliferative activity of an extract and individual compounds were investigated in immortalized human non-tumorigenic keratinocytes (HaCaT). A MeOH extract from aerial parts afforded several flavonoids, including luteolin and apigenin glycosides (2-5 and 9) and catechin (1), two procyanidin fractions, and the phenolic glucosides picein (6), triandrin (7), and salicortin (8). In an adenosine triphosphate assay, the MeOH extract reduced cell viability by approximately 60 % at a concentration of 100 µg/mL. Cell proliferation was assessed with a BrdU incorporation ELISA assay. The extract inhibited proliferation of HaCaT cells in a concentration-dependent manner, with approximately 50 % inhibition at 100 µg/mL. In time-lapse assays, the extract showed distinct inhibitory effects on cell migration at concentrations of 12.5, 25, and 50 µg/mL. The activity of selected constituents was also determined. Luteolin-7-O-β-glucuronide (3) significantly inhibited cell proliferation at concentrations of 10 and 50 µM. In contrast, luteolin-7-O-β-glucopyranoside (2) and a procyanidin fraction (P1) had only weak effects, while picein (6) and salicortin (8) did not affect cell proliferation. Luteolin-7-O-β-glucuronide (10 µM) and, to a lesser extent, the procyanidin fraction (10 µg/mL) also inhibited cell migration.

Exposure to sub-10 nm particles emitted from a biodiesel-fueled diesel engine: In vitro toxicity and inflammatory potential

ObjectivesThe inflammatory effects of organic sub-10nm particles generated and emitted from a diesel engine fueled with a biodiesel and a commercial diesel oil are analyzed in this paper. Diesel combustion is the major sources of ultrafine particles (UFP) in the environment, particularly in urbanized areas. In the last years, there is an increasing use of biomass-derived fuels because they are a renewable source of energy that may mitigate climate change through the reduction of net CO2 with respect to conventional fossil fuels. Although there is a general agreement on biofuels ability to reduce conventional pollutants, new and potentially harmful pollutants can be formed during biofuel combustion. In particular, the emission of sub-10nm particles is strongly increased with respect to that of larger soot particles. MethodsOrganic sub-10nm particles are separated from larger sizes particulate matter by collection in water suspension for toxicological and inflammatory tests. After exposure to sub-10nm particles, the effects on proliferation, apoptosis and secretion of cytokines, chemokines and growth factors networks production is analyzed in immortalized non-tumorigenic human dermal keratinocyte cell line (HaCaT) and human alveolar epithelial-like cells (A549). Results and conclusionNanoparticles exert different cytotoxic effects in the two cell lines, suggesting that the dermal way of exposure is more sensitive than the inhalant way. These differences are most evident in the secretion of pro-inflammatory, angiogenic and proliferative cytokines and chemokines whose expression is more finely modulated in HaCaT cells compared to A-549 cells. Considering the size of these particles, it is important to promote the culture of prevention also for the dermal way in particularly exposed workers.

Zinc oxide nanoparticles-induced epigenetic change and G2/M arrest are associated with apoptosis in human epidermal keratinocytes.

As an engineered nanomaterial, zinc oxide nanoparticles (ZnO NPs) are used frequently in biological applications and can make contact with human skin. Here, we systematically investigated the effects of ZnO NPs on non-tumorigenic human epidermal keratinocytes, which were used as a test model for this in vitro study, at the epigenetic and molecular levels. Our results showed that ZnO NPs induced cell cycle arrest at the G2/M checkpoint before the viability of human epidermal keratinocytes was reduced, which was associated with the chromatin changes at the epigenetic level, including increased methylation of histone H3K9 and decreased acetylation of histone H4K5 accompanied by chromatin condensation at 24 hours. The mRNA expression of the methyltransferase genes G9a and GLP was also increased upon treatment with ZnO NPs, and the acetyltransferase genes GCN5, P300, and CBP were downregulated. Reactive oxygen species were found to be more abundant after treatment with ZnO NPs for 6 hours, and DNA damage was observed at 24 hours. Transmission electron microscopy and flow cytometry confirmed that ZnO NPs were absorbed into the cell when they were added to the medium. Apoptotic human epidermal keratinocytes were detected, and the expression of the proapoptotic genes Bax, Noxa, and Puma increased significantly, while the expression of the antiapoptotic gene Bcl-xl decreased 24 hours after exposure to ZnO NPs. These findings suggest that the ZnO NPs induced cell cycle arrest at G2/M, which was associated with epigenetic changes and accompanied by p53-Bax mitochondrial pathway-mediated apoptosis.

Effect of fucoidan from Turbinaria conoides on human lung adenocarcinoma epithelial (A549) cells

Fucoidan was purified from seaweed, Turbinaria conoides. Isolated fragments were characterized with NMR (13C, 1H), Gas Chromatography–Mass Spectronomy (GC–MS) and HPLC analysis. The autohydrolysate of fucoidans consisted of sulfated fuco-oligosaccharides having the backbone of α-(1, 3)-linked fuco-pyranose derivatives and minor components of galactose, glucose, mannose and xylose sugars. Fucoidan induced a dose-dependent reduction in cell survival of lung cancer A549 cells by MTT assay (GI50, 75μg/mL). However, it was not cytotoxic to a non-tumorigenic human keratinocyte cell line of skin tissue (HaCaT) (GI50>1.0mg/mL). The apoptotic cells in fucoidan-treated A549 cells were visualized by laser confocal microscopy and cell cycle analysis showed induction of G0/G1 phase arrest of the cell progression cycle. Further, CFSE labeling and flow cytometry highlighted that fucoidan significantly (P<0.05) inhibited the proliferation rate of A549 cells by up to 2-fold compared with the control cells. It is concluded that fucoidan has the potential to act as an anti-proliferative agent on lung carcinoma (A549) cells.

TG-interacting factor mediates arsenic-induced malignant transformation of keratinocytes via c-Src/EGFR/AKT/FOXO3A and redox signalings.

Inorganic arsenic is well known as a carcinogen in human beings. Chronic exposure to inorganic arsenic increases risks of developing some cancers and non-carcinogenic diseases, such as skin lesions in humans. However, the modes of action are not well elucidated. In the present study, HaCaT cells, an immortalized non-tumorigenic human keratinocyte, were continuously exposed to low-dose trivalent arsenic (arsenic trioxide, 0.1 and 0.2 μM) for at least 4 weeks. We proved that low-dose arsenic could stimulate malignant transformation of HaCaT cells, including increase of cellular proliferation, epithelial-to-mesenchymal transition markers alteration, matrix metalloproteinases activation, invadopodia formation, migration/invasion activities, and anchorage-independent growth. Surprisingly, low-dose arsenic could also transcriptionally increase TG-interacting factor (TGIF) expression via c-Src/EGFR/AKT/FOXO3A signaling involving superoxide production from NADPH oxidase. Moreover, stable overexpression of TGIF could also induce malignant transformation of HaCaT cells. Knockdown of TGIF with its specific shRNA abolished the arsenic-induced effects. Taken together, we suggest that TGIF plays an important role in low-dose arsenic-induced malignant transformation of HaCaT cells, which is regulated by c-Src/EGFR/AKT/FOXO3A pathway and redox signaling.

Cytotoxic activity of aquatic fungus Pseudohalonectria lignicola on two different human cell lines

Water covers nearly three-quarters of the earth's surface and host a diversity of unexplored microbes. Considerable researches have been done to expose the bioactive potential of free living and symbiotic marine and fresh water microorganisms, many of them have proved to be potent producers of bioactive secondary metabolites. Beside bacteria and algae, fungi represent a comprehensive and promising source of novel anticancer, antibacterial, anti-inflammatory, antiviral and cytotoxic agents. Therefore the limnetic fungus Pseudohalonectria lignicola, first described by Minoura and Muroi in 1978, was investigated in more detail. Previous experiments exhibited antibiotic, antifungal and nematicidal properties of P. lignicola. All studies about this fungus were done on a isolated P. lignicola strain from Baltic Sea. In search of new characteristics of this strain we generated 10 HPLC fractions from the methanolic extract and determined cell viability by treatment of human bladder cancer cell line 5637 and nontumorigenic human HaCaT keratinocytes using MTT assay. Fraction 4, 5 and 6 showed strong cytotoxic effects with IC50 values of 18.7 µg/ml, 17.9 µg/ml and 17.3 µg/ml on 5637 cell line. Fraction 3 and 6 were also cytotoxic for HaCaT cells. Isolation and structure elucidation of active fractions and pure substances are currently underway. The natural substance nectriapyrone has already been identified. Our generated fungus P. lignicola shows both antibacterial and cytotoxic effects that is why it could be a promising candidate for cancer treatment or for usage as antibiotic agent.

Abstract 1598: Grainyhead-like 2 (GRHL2) regulates the expression of forkhead box transcription factor M1 (FoxM1) in human oral cancer cells and determines tumorigenicity

Abstract GRHL2 is a proto-oncogene that regulates epithelial proliferation and differentiation through transcriptional regulation of large number of target genes. The current study was performed to determine its role in oral carcinogenesis through regulation of forkhead box 1 (FoxM1), which is a known oncogene for many solid tumors. GRHL2 expression was highly elevated in oral squamous cell carcinoma (OSCC) tissues compared with normal oral epithelium (NHOM), and its protein expression is localized in the proliferative cells at the outer border of invasive islands of OSCC. In cultured OSCCs, GRHL2 and FoxM1 levels were both highly upregulated compared with normal human oral keratinocytes (NHOK), supporting their roles in oral carcinogenesis. When GRHL2 was knocked down in SCC4 cells by RNA interference, the cells have lost their proliferative capacity and colony formation when plated at low density. GRHL2 knockdown also resulted in marked loss of tumor spheroid formation, which is hallmark of cancer self-renewal. GRHL2 knockdown also led to loss of tumorigenic ability of cells upon xenograft transplantation in immunocompromised mice. GRHL2 appeared to determine the epithelial phenotype in OSCC cells. GRHL2 knockdown led to loss of E-cadherin and miR-200 family genes, which are epithelial markers. These data indicate that GRHL2 is required to maintain the transformed phenotype in OSCC. We also noted that GRHL2 knockdown led to drastic loss of FoxM1 expression, suggesting that GRHL2 may regulate FoxM1. To test this possibility, we performed in vivo binding assay by chromatin immunoprecipitation (ChIP), which showed GRHL2 binding at the proximal promoter of FoxM1. Luciferase reporter plasmid under FoxM1 promoter showed that GRHL2 directly regulates the gene promoter activity. FoxM1 knockdown resulted in loss of OSCC proliferation and tumor spheroid formation, while its overexpression conferred transformed phenotype in non-tumorigenic human oral keratinocytes (HOKs). Therefore, our data indicate that GRHL2 is required for the maintenance of transformed phenotype and that it plays a role in oral carcinogenesis through regulation of FoxM1. This study was supported in part by the grants from NIDCR/NIH (DE18295 and DE18959) and Jack Weichman Endowed Fund. Note: This abstract was not presented at the meeting. Citation Format: Wei Chen, Jin-Kyu Yi, Ki-Hyuk Shin, Reuben Kim, Shebli Mehrazarin, No-Hee Park, Mo K. Kang. Grainyhead-like 2 (GRHL2) regulates the expression of forkhead box transcription factor M1 (FoxM1) in human oral cancer cells and determines tumorigenicity. [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 1598. doi:10.1158/1538-7445.AM2014-1598

A Green Approach Toward Quinoxalines and Bis -Quinoxalines and Their Biological Evaluation Against A431, Human Skin Cancer Cell Lines

The objective of this study was to develop a practical green procedure to synthesize quinoxalines and bis-quinoxalines and evaluate their inhibitory effects on the viability of A431 human epidermoid carcinoma cells. A series of quinoxaline and bis-quinoxaline derivatives have been designed and synthesized following a microwave-assisted and bismuth nitrate-catalyzed eco-friendly route. A detailed comparison has been made between microwave-induced protocol with the reactions occurred at room temperature. The structure of the compounds have been elucidated by various spectroscopic methods and finally confirmed by x-ray crystallographic analyses. Two quinoxaline derivatives, compounds 6 and 12 have demonstrated inhibitory effects on the viability of A431 human epidermoid carcinoma cells when compared with HaCaT nontumorigenic human keratinocyte cells. Notably, compound 6 inhibits Stat3 phosphorylation/activation in A431 skin cancer cells.

Enhanced constitutive invasion activity in human nontumorigenic keratinocytes exposed to a low level of barium for a long time

We have recently demonstrated that exposure to barium for a short time (≤4 days) and at a low level (5 µM = 687 µg/L) promotes invasion of human nontumorigenic HaCaT cells, which have characteristics similar to those of normal keratinocytes, suggesting that exposure to barium for a short time enhances malignant characteristics. Here we examined the effect of exposure to low level of barium for a long time, a condition mimicking the exposure to barium through well water, on malignant characteristics of HaCaT keratinocytes. Constitutive invasion activity, focal adhesion kinase (FAK) protein expression and activity, and matrix metalloproteinase 14 (MMP14) protein expression in primary cultured normal human epidermal keratinocytes, HaCaT keratinocytes, and HSC5 and A431 human squamous cell carcinoma cells were augmented following an increase in malignancy grade of the cells. Constitutive invasion activity, FAK phosphorylation, and MMP14 expression levels of HaCaT keratinocytes after treatment with 5 µM barium for 4 months were significantly higher than those of control untreated HaCaT keratinocytes. Taken together, our results suggest that exposure to a low level of barium for a long time enhances constitutive malignant characteristics of HaCaT keratinocytes via regulatory molecules (FAK and MMP14) for invasion.