Cultured Carcinoma Cells Research Articles

New evidences on the regulation of SF-1 expression by POD1/TCF21 in adrenocortical tumor cells

OBJECTIVES:Transcription Factor 21 represses steroidogenic factor 1, a nuclear receptor required for gonadal development, sex determination and the regulation of adrenogonadal steroidogenesis. The aim of this study was to investigate whether silencing or overexpression of the gene Transcription Factor 21 could modulate the gene and protein expression of steroidogenic factor 1 in adrenocortical tumors.METHODS:We analyzed the gene expression of steroidogenic factor 1 using qPCR after silencing endogenous Transcription Factor 21 in pediatric adrenal adenoma-T7 cells through small interfering RNA. In addition, using overexpression of Transcription Factor 21 in human adrenocortical carcinoma cells, we analyzed the protein expression of steroidogenic factor 1 using Western blotting.RESULTS:Transcription Factor 21 knockdown increased the mRNA expression of steroidogenic factor 1 by 5.97-fold in pediatric adrenal adenoma-T7 cells. Additionally, Transcription Factor 21 overexpression inhibited the protein expression of steroidogenic factor 1 by 0.41-fold and 0.64-fold in two different adult adrenocortical carcinoma cell cultures, H295R and T36, respectively.CONCLUSIONS:Transcription Factor 21 is downregulated in adrenocortical carcinoma cells. Taken together, these findings support the hypothesis that Transcription Factor 21 is a regulator of steroidogenic factor 1 and is a tumor suppressor gene in pediatric and adult adrenocortical tumors.

Methylation of promoter of RBL1 enhances the radioresistance of three dimensional cultured carcinoma cells.

Three dimensional (3D) culture in vitro is a new cell culture model that more closely mimics the physiology features of the in vivo environment and is being used widely in the field of medical and biological research. It has been demonstrated that cancer cells cultured in 3D matrices are more radioresistant compared with cells in monolayer (2D). However, the mechanisms causing this difference remain largely unclear. Here we found that the cell cycle distribution and expression of cell cycle regulation genes in 3D A549 cells are different from the 2D. The higher levels of the promotor methylation of cell cycle regulation genes such as RBL1 were observed in 3D A549 cells compared with cells in 2D. The treatments of irradiation or 5-Aza-CdR activated the demethylation of RBL1 promotor and resulted in the increased expression of RBL1 only in 3D A549 cells. Inhibition of RBL1 enhanced the radioresistance and decreased the G2/M phase arrest induced by irradiation in 2D A549 and MCF7 cells. Overexpression of RBL1 sensitized 3D cultured A549 and MCF7 cells to irradiation. Taken together, to our knowledge, it is the first time to revealthat the low expression of RBL1 due to itself promotor methylation in 3D cells enhances the radioresistance. Our finding sheds a new light on understanding the features of the 3D cultured cell model and its application in basic research into cancer radiotherapy and medcine development.

Adipose-Derived Stem Cells Enhance Cancer Stem Cell Property and Tumor Formation Capacity in Lewis Lung Carcinoma Cells Through an Interleukin-6 Paracrine Circuit.

Adipose-derived stem cells (ADSCs) are multipotent cells that have attracted much recent attention and emerged as therapeutic approaches in several medical fields. Although current knowledge of the biological impacts of ADSCs in cancer research is greatly improved, the underlying effects of ADSCs in tumor development remain controversial and cause the safety concerns in clinical utilization. Hence, we isolated primary ADSCs from the abdominal fat of mice and conducted interaction of ADSCs with Lewis lung carcinoma cells in culture and in mice to investigate the impacts of ADSCs on tumor development. Cytokine array and neutralizing antibody were further utilized to identify the key regulator and downstream signaling pathway. In this study, we demonstrated that ADSCs enhance the malignant characteristics of LLC1 cells, including cell growth ability and especially cancer stem cell property. ADSCs were then identified to promote tumor formation and growth in mice. We further determined that ADSC interaction with LLC1 cells stimulates increased secretion of interleukin-6 mainly from ADSCs, which then act in a paracrine manner on LLC1 cells to enhance their malignant characteristics. Interleukin-6 was also identified to regulate genes related to cell proliferation and cancer stem cell, as well as to activate JAK2/STAT3, a predominant interleukin-6-activated pathway, in LLC1 cells. Collectively, we demonstrated that ADSCs play a pro-malignant role in tumor development of Lewis lung carcinoma cells by particularly promoting cancer stem cell property through interleukin-6 paracrine circuit, which is important for safety considerations regarding the clinical application of ADSCs.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Ascorbate availability affects tumor implantation-take rate and increases tumor rejection in Gulo-/- mice.

In solid tumors, HIF1 upregulates the expression of hundreds of genes involved in cell survival, tumor growth, and adaptation to the hypoxic microenvironment. HIF1 stabilization and activity are suppressed by prolyl and asparagine hydroxylases, which require oxygen as a substrate and ascorbate as a cofactor. This has led us to hypothesize that intracellular ascorbate availability could modify the hypoxic HIF1 response and influence tumor growth. In this study, we investigated the effect of variable intracellular ascorbate levels on HIF1 induction in cancer cells in vitro, and on tumor-take rate and growth in the Gulo−/− mouse. These mice depend on dietary ascorbate, and were supplemented with 3,300 mg/L, 330 mg/L, or 33 mg/L ascorbate in their drinking water, resulting in saturating, medium, or low plasma and tissue ascorbate levels, respectively. In Lewis lung carcinoma cells (LL/2) in culture, optimal ascorbate supplementation reduced HIF1 accumulation under physiological but not pathological hypoxia. LL/2, B16-F10 melanoma, or CMT-93 colorectal cancer cells were implanted subcutaneously into Gulo−/− mice at a range of cell inocula. Establishment of B16-F10 tumors in mice supplemented with 3,300 mg/L ascorbate required an increased number of cancer cells to initiate tumor growth compared with the number of cells required in mice on suboptimal ascorbate intake. Elevated ascorbate intake was also associated with decreased tumor ascorbate levels and a reduction in HIF1α expression and transcriptional activity. Following initial growth, all CMT-93 tumors regressed spontaneously, but mice supplemented with 33 mg/L ascorbate had lower plasma ascorbate levels and grew larger tumors than optimally supplemented mice. The data from this study indicate that improved ascorbate intake is consistent with increased intracellular ascorbate levels, reduced HIF1 activity and reduced tumor initiation and growth, and this may be advantageous in the management of cancer.

Correlative NAD(P)H-FLIM and oxygen sensing-PLIM for metabolic mapping.

Cellular responses to oxygen tension have been studied extensively. Oxygen tension can be determined by considering the phosphorescence lifetime of a phosphorescence sensor. The simultaneous usage of FLIM of coenzymes as NAD(P)H and FAD(+) and PLIM of oxygen sensors could provide information about correlation of metabolic pathways and oxygen tension. We investigated correlative NAD(P)H-FLIM and oxygen sensing-PLIM for simultaneously analyzing cell metabolism and oxygen tension. Cell metabolism and pO2 were observed under different hypoxic conditions in squamous carcinoma cell cultures and in complex ex vivo systems. Increased hypoxia induced an increase of the phosphorescence lifetime of Ru(BPY)3 and in most cases a decrease in the lifetime of NAD(P)H which is in agreement to the expected decrease of the protein-bound NAD(P)H during hypoxia. Oxygen was modulated directly in the mitochondrial membrane. Blocking of complex III and accumulation of oxygen could be observed by both the decrease of the phosphorescence lifetime of Ru(BPY)3 and a reduction of the lifetime of NAD(P)H which was a clear indication of acute changes in the redox state of the cells. For the first time simultaneous FLIM/PLIM has been shown to be able to visualize intracellular oxygen tension together with a change from oxidative to glycolytic phenotype.

Antiproliferative activity of ruthenium and osmium clusters with phosphine ligands

New ruthenium and osmium carbonyl clusters with 1,3,5-triaza-7-phosphatricyclo-[3.3.1.13.7]decane and 3,7-diacetyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane ligands were synthesized using Me3NO as an initiator. The data on antiproliferative activity of new compounds against ovarian carcinoma cell cultures A2780 (cisplatin-sensitive) and A2780cisR (cisplatin-resistant) are reported. The dependence of cytotoxicity on the number of phosphine ligands was demonstrated.

Characterization and cytotoxicity studies of thiol-modified polystyrene microbeads doped with [{Mo6X8}(NO3)6]2-(X = Cl, Br, I)

Halide octahedral molybdenum clusters [(Mo6X8)L6]n- possess luminescence properties that are highly promising for biological applications. These properties are rather dependent on the nature of both the inner ligands X (i.e. Cl, Br, or I) and the apical organic or inorganic ligands L. Herein, the luminescence properties and the toxicity of thiol-modified polystyrene microbeads (PS-SH) doped with [(Mo6X8)(NO3)6]2- (X=Cl, Br, I) were studied and evaluated using human epidermoid larynx carcinoma (Hep2) cell cultures. According to our data, the photoluminescence quantum yield of (Mo6I8)@PS-SH is significantly higher (0.04) than that of (Mo6Cl8)@PS-SH (6Br8)@PS-SH (6X8)@PS-SH showed that all three types of doped microbeads had no significant effect on the viability and proliferation of the cells.

Multifunctional Pt(II) Reagents: Covalent Modifications of Pt Complexes Enable Diverse Structural Variation and In-Cell Detection.

To enhance the functionality of Pt-based reagents, several strategies have been developed that utilize Pt compounds modified with small, reactive handles. This Account encapsulates work done by us and other groups regarding the use of Pt(II) compounds with reactive handles for subsequent elaboration with fluorophores or other functional moieties. Described strategies include the incorporation of substituents for well-known condensation or nucleophilic displacement-type reactions and their use, for example, to tether spectroscopic handles to Pt reagents for in vivo investigation. Other chief uses of displacement-type reactions have included tethering various small molecules exhibiting pharmacological activity directly to Pt, thus adding synergistic effects. Click chemistry-based ligation techniques have also been applied, primarily with azide- and alkyne-appended Pt complexes. Orthogonally reactive click chemistry reactions have proven invaluable when more traditional nucleophilic displacement reactions induce side-reactivity with the Pt center or when systematic functionalization of a larger number of Pt complexes is desired. Additionally, a diverse assortment of Pt-fluorophore conjugates have been tethered via click chemistry conjugation. In addition to providing a convenient synthetic path for diversifying Pt compounds, the use of click-capable Pt complexes has proved a powerful strategy for postbinding covalent modification and detection with fluorescent probes. This strategy bypasses undesirable influences of the fluorophore camouflaged as reactivity due to Pt that may be present when detecting preattached Pt-fluorophore conjugates. Using postbinding strategies, Pt reagent distributions in HeLa and lung carcinoma (NCI-H460) cell cultures were observed with two different azide-modified Pt compounds, a monofunctional Pt(II)-acridine type and a difunctional Pt(II)-neutral complex. In addition, cellular distribution was observed with an alkyne-appended difunctional Pt(II)-neutral complex analogous in structure to the aforementioned difunctional azide-Pt(II) reagent. In all cases, significant accumulation of Pt in the nucleolus of cells was observed, in addition to broader localization in the nucleus and cytoplasm of the cell. Using the same strategy of postbinding click modification with fluorescent probes, Pt adducts were detected and roughly quantified on rRNA and tRNA from Pt-treated Saccharomyces cerevisiae; rRNA adducts were found to be relatively long-lived and not targeted for immediate degradation. Finally, the utility and feasibility of the alkyne-appended Pt(II) compound has been further demonstrated with a turn-on fluorophore, dansyl azide, in fluorescent detection of DNA in vitro. In all, these modifications utilizing reactive handles have allowed for the diversification of new Pt reagents, as well as providing cellular localization information on the modified Pt compounds.

Abstract A104: Development of pathophysiologically relevant in vitro tumor models: Cytotoxicity of anticancer drugs in two dimensional and three dimensional cell line cultures versus primary colorectal tumor cells

Abstract Chemotherapy is an effective modality for cancer therapy. However, treatment of solid tumors is hampered by several factors, such as development of acquired resistance, nonspecific drug effects, etc. Moreover, high attrition rate of anticancer drugs is an additional clinical hurdle often encountered in cancer patients. One of the potential ways to overcome these issues is to use in vitro tumor models that reliably capture in vivo tumor conditions for effective drug selection in the preclinical stages. Although two-dimensional (2D) cultures of cancer cells are widely used for drug screening and target validation; they are less predictive in vitro tumor models than three-dimensional (3D) cultures and primary cultures of patient-derived tumor cells. Cytotoxic drugs, nanoparticles, and antitumor antibodies display altered activity in 2D, 3D, and primary cancer cell cultures, potentially due to cellular and molecular differences in these culture types. Recently, the use of multicellular 3D spheroid cultures of cancer cells have gained considerable attention among researchers as more predictive in vitro tumor models to study the effects of tumor microenvironment on cancer cell response to drugs. In addition to 3D cultures, patient-derived tumor cells are also becoming increasingly popular in drug research for their relative closeness to tumors. It is expected that drugs characterized using these pathophysiologically relevant in vitro tumor models will improve their translation to higher disease models, and significantly reduce the cost associated with failure of drugs in the clinical trials. In this study, we compare the cytotoxic effects of 12 standard anticancer drugs in 2D and 3D cultures of colorectal carcinoma (CRC) cells to >500 primary CRC cultures. Furthermore, using primary cultures of patient-derived ovarian, breast, and lung cancer cells, we present the potential use of primary tumor cells as in vitro predictive model for chemotherapy. Our data indicates that compared to 2D cultures, 3D cultures of CRC cells better reflect the drug effects seen in primary CRC cultures. Our cytotoxicity data from primary cultures also show how the drug sensitivity profiles differ across histologically different solid tumors types. Acknowledgements This work was supported by grants from the Czech Ministry of Education, Youth and Sports (CZ.1.07/2.3.00/30.0041 to V.D.; LO1304 to M.H.), the Czech Ministry of Health (NT14282 to M.H.), and the Technological Agency of the Czech Republic (TE01020028 to M.H.). Citation Format: Viswanath Das, Marta Zbozinkova, Martin Holoubek, Petr Dzubak, Marian Hajduch. Development of pathophysiologically relevant in vitro tumor models: Cytotoxicity of anticancer drugs in two dimensional and three dimensional cell line cultures versus primary colorectal tumor cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A104.

Prostaglandins induce early growth response 1 transcription factor mediated microsomal prostaglandin E2 synthase up-regulation for colorectal cancer progression.

Cyclooxygenase2 (COX2) has been associated with cell growth, invasiveness, tumor progression and metastasis of colorectal carcinomas. However, the downstream prostaglandin (PG)-PG receptor pathway involved in these effects is poorly characterized.We studied the PG-pathway in gene expression databases and we found that PTGS2 (prostaglandin G/H synthase and cyclooxygenase) and PTGES (prostaglandin E synthase) are co-expressed in human colorectal tumors. Moreover, we detected that COX2 and microsomal Prostaglandin E2 synthase 1 (mPGES1) proteins are both up-regulated in colorectal human tumor biopsies.Using colon carcinoma cell cultures we found that COX2 overexpression significantly increased mPGES1 mRNA and protein. This up-regulation was due to an increase in early growth response 1 (EGR1) levels and its transcriptional activity. EGR1 was induced by COX2-generated PGF2α. A PGF2α receptor antagonist, or EGR1 silencing, inhibited the mPGES1 induction by COX2 overexpression. Moreover, using immunodeficient mice, we also demonstrated that both COX2- and mPGES1-overexpressing carcinoma cells were more efficient forming tumors.Our results describe for the first time the molecular pathway correlating PTGS2 and PTGES in colon cancer progression. We demonstrated that in this pathway mPGES1 is induced by COX2 overexpression, via autocrine PGs release, likely PGF2α, through an EGR1-dependent mechanism. This signaling provides a molecular explanation to PTGS2 and PTGES association and contribute to colon cancer advance, pointing out novel potential therapeutic targets in this oncological context.

Antimetastatic effect of fluvastatin on breast and hepatocellular carcinoma cells in relation to SGK1 and NDRG1 genes.

Metastasis occurs due to migration of the cells from primary tumor toward other tissues by gaining invasive properties. Since metastatic invasion shows a strong resistance against conventional cancer treatments, the studies on this issue have been focused. Within this context, inhibition of migration and determination of the relationships at the gene level will contribute to treatment of metastatic cancer cases. We have aimed to demonstrate the impact of TGF-β1 and fluvastatin on human breast cancer (MCF-7) and human hepatocellular carcinoma (Hep3B) cell cultures via Real-Time Cell Analyzer (RTCA) and to test the expression levels of some genes (NDRG1, SGK1, TWIST1, AMPKA2) and to compare their gene expression levels according to RTCA results. Both of cell series were applied TGF-β1 and combinations of TGF-β1/fluvastatin. Primer and probes were synthesized using Universal Probe Library (UPL, Roche) software, and expression levels of genes were tested via qPCR using the device LightCycler 480 II (Roche). Consequently, fluvastatin dose-dependently inhibited migration induced by TGF-β1 in both groups. This inhibition was accompanied by low level of SGK1 messenger RNA (mRNA) and high levels of NDRG1 and AMPKA2 mRNA. Thus, we conclude that fluvastatin plays an important role in reducing resistance to chemotherapeutics and preventing metastasis.

Abstract 5533: A local chemotherapy with hyaluronan-cisplatin conjugate significantly attenuates growth of lung adenocarcinoma xenografts in mouse model

Abstract Nanoparticle-based chemotherapy, while promising, remains clinically unsuccessful, mainly due to a lack of targeted delivery methods of the therapeutics into cancer tissues and the side effect of chemotherapeutics. We have demonstrated that the nanoparticle formulation with hyaluronan-cisplatin conjugates (HylaPlat) is suggested to be an effective chemotherapeutic delivery method in breast cancer mouse models (Cai, et al., J Surg Res., 2008; Cai, et al., Ther Deliv, 2011). The objectives of the present study were to examine the growth inhibition efficacy of HylaPlat formulation on lung adenocarcinoma cells in cell culture and to examine the therapeutic efficiency of the local administration of HylaPlat on lung adenocarcinoma in mice. Effect of the HylaPlat on the growth of lung carcinoma was evaluated using Lewis Lung carcinoma (LLC) cells in 2D culture and 3D spheroid cell culture, as well as, orthotopic autografts in C57BL/6 mice lungs. Cell culture studies clarified that the HylaPlat effectively attenuated cell growth in 2D and 3D spheroid culture with IC50 of 0.35μM and 1.35 μM, respectively; whereas control cisplatin-dependent growth inhibition was achieved with several fold higher concentrations (IC50 1.64 and 3.6 μM, respectively). The 3D spheroid cell culture study also revealed that the treatment with HylaPlat induced apoptosis in the cells located on the peripheral area of the spheroid first, this effect lasted for several days. A single intratracheal administration of 7.5mg/kg HylaPlat (1mg cisplatin equivalent/kg) seven days after LLC cell inoculation almost completely inhibited growth of LLC autografts in the mouse lungs. Histological analysis of dissected lungs revealed that a small number of microscopic tumor nodules were detected in the treated mouse lungs, whereas several large tumors were detected in the untreated control mouse lungs. Normal lung architectures of the trachea, bronchioles and alveoli were maintained in the treated mice although small inflammation spots can be detected at peripheral areas. Apoptotic index was significantly higher in the treated tumors than PBS treated control tumors, suggesting that cisplatin was successfully delivered to the tumor tissues by nanoparticle formulated HylaPlat and caused apoptosis of tumor cells. The pharmacokinetics of cisplatin 14 days after intratracheal administration of HylaPlat is under investigation. Taken together, the current study suggests that an intratracheal administration of HylaPlat nanoparticles offer an effective strategy for lung cancer treatment. This work was supported by the Kansas State University Johnson Cancer Research Center, Kansas Bioscience Authority Collaborative Cancer Research grant, 1R01CA173292 (LF) and American Cancer Society RSG-08-133-01-CDD (LF). Citation Format: Susumu Ishiguro, Deepthi Uppalapati, Shuang Cai, Katie Turner, Jacob Hodge, Laird Forest, Masaaki Tamura. A local chemotherapy with hyaluronan-cisplatin conjugate significantly attenuates growth of lung adenocarcinoma xenografts in mouse model. [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 5533. doi:10.1158/1538-7445.AM2015-5533

Abstract 1699: Differential aggregation rates and therapeutic response of pancreatic cancer cell lines to sigma-2 receptor activation in 3D culture

Abstract Pancreatic cancer is one of the most deadly forms of cancer, with overall 5-year survival rates under 20%. The only cure for this type of cancer is surgical, however this option is rarely available due in part to detection that is typically late in the progression of the disease. The aim of this study was to understand the differences in progression between L3.6, PANC-1, and M1A-PaCa-2 cells as representatives of varied levels of metastatic character through aggregation analysis in 3D cell culture. It is well established that 3D culture more closely mimics the in vivo condition, as the cells adhere to each other forming “microtumors” as opposed to being attached to a plastic substratum as is the case with standard 2D cell culture. We also examined the effectiveness of a novel target for chemotherapeutic intervention in these cell lines in 3D culture. The sigma-2 receptor is highly upregulated in rapidly proliferating cancer tissues as compared to non-cancerous tissue, and induces apoptosis when activated. We previously reported the ability of the sigma-2 receptor partial agonist CM572 (sigma-2 Ki = 14.7 nM) to induce apoptosis in PANC-1 pancreatic carcinoma cells in standard 2D culture (Nicholson et al., Proc. Amer. Assoc. Cancer Res. 74: #3237, 2014). Here, we measured the aggregation rate and sigma-2 agonist sensitivity of L3.6, a highly metastatic cell line, in comparison to M1A-PaCa-2 and PANC-1 cells, which are both significantly less aggressive. The sigma-2 receptor agonist siramesine (Ki = 0.12nM) was found to induce cell death in 3D tumor cell culture in all three pancreatic cancer cell lines. However, the potency was 30-50% of the potency observed in traditional 2D cultured cells. Interestingly, in 3D culture, L3.6 and PANC-1 cells both exhibited ∼45% of the maximal cell death that was achieved in 2D culture using the same dose of siramesine, and M1A-PaCa-2 cells exhibited ∼25% of the maximal 2D toxicity at this dose. Additionally, both siramesine and CM572 were able to slow aggregation of the pancreatic cancer cells. This data indicates that the sigma-2 receptor may provide a novel target for therapeutic intervention in pancreatic cancer, both for slowing progression of the disease and as a chemotherapeutic target. The data also indicate that cell-cell contact in 3D culture either modifies the sigma-2 receptor signaling process or that cells in 3D culture present more of a barrier for drug access to the tumor cells. Further studies are needed to examine these possibilities. Citation Format: Timothy Chou, Michaela Jacobs, Hilary Nicholson, Wayne D. Bowen. Differential aggregation rates and therapeutic response of pancreatic cancer cell lines to sigma-2 receptor activation in 3D culture. [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 1699. doi:10.1158/1538-7445.AM2015-1699

Abstract 3248: Establishment and characterization of primary penile carcinoma cell culture versus xenograft by using transcriptome analysis

Abstract Background: The establishment of tumor cell lines in penile carcinoma (PeCa), a rare and high morbidity disease, is essential to conduct functional genomic and epigenomic studies. However, penile cancer cell line is not commercially available. In addition, xenograft in immunodeficient mouse is a valuable model between in vitro testing and pre-clinical trials. Aim: This study aimed to establish and characterize PeCa primary cultures and xenografts by using large-scale gene expression analysis. Methods: Primary tumor cultures were established from one untreated verrucous PeCa sample (HPV- negative). Tumor tissue was minced and digested with 0.2% trypsin for 30 minutes at 37°C. After extensively washing with 3 KSFM (GIBCO):1 DF12 (GIBCO). After passage 5 (36 days), two cell cultures with distinct morphologies were isolated and implanted subcutaneously on the right (epithelial cells) and left (fibroblast-like cells) flanks of BALB/C nude mouse. Analyses of AE1AE3(ABCAM), Vimentin (ABCAM), Desmin (DAKO), Myogenine (SIGMA), HHF35 (DAKO), 1A3 (SIGMA), 34βE12 (ABCAM) and CD44 (DAKO) by immunohistochemistry and ALDH (ABCAM) by flow cytometry were performed in xenograft tissues. Total RNA was extracted from cell cultures and xenograft tissues in duplicate and evaluated for gene expression by using Human Transciptome Array (HTA) 2.0 (Affymetrix). Expression Console (Affymetrix), SAM, hierarchical clustering analysis, Ingenuity Pathway Analysis (IPA). Results: After 5 passages of verrucous PeCa cells, distinct epithelial and fibroblast-like cell morphologies were observed. After four weeks of inoculation of both cell types into right and left flanks of nude mouse, tumor growth was observed in both flanks. Both tumor tissues obtained from the patient and xenograft showed positive immunostaining for epithelial cell markers (AE1AE3, 5-10% of cells and high molecular weight cytokeratin 34βE12, 15% of cells), for mesenchymal cells (vimentin, 95%; myogenin, 95%; desmin, 80-90%; HHF35, 10%; 1A4, 95%) and also for stem cells (CD44, 5% of cells). ALDH activity was evaluated by flow cytometry and high ALDH activity was detected in vitro and in vivo cells. Preliminary results from transcriptome analysis showed 485 differentially expressed genes between primary tumor and Pre Xenograft passage 1; 502 genes in primary tumor and Pre Xenograft passage 5; 468 genes in verrucous PeCa sample vs xenografts passage 1 and 2; 402 genes in verrucous PeCa sample vs xenograft passage 3. It was verified upregulation of STAT3, IFNg, KRT14 and IFNA2 genes as well as genes involved in ROCK2 signaling networks. Conclusions: We successfully established a primary tumor cell culture and xenograft derived from a verrucous penile carcinoma. Our data also support that despite cell cultures showed different morphologies compared to primary tumor, they present genotypic and phenotypic features similar to the tissue of origin. Citation Format: Juan José Augusto Moyano Muñoz, Sandra A. Drigo, Tiago Goss, Hellen Kuasne, Fabio Marchi, Gustavo Guimaraes, Ademar Lopes, Cristovam S. Neto, Silvia R. Rogatto. Establishment and characterization of primary penile carcinoma cell culture versus xenograft by using transcriptome analysis. [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 3248. doi:10.1158/1538-7445.AM2015-3248

Abstract 2224: Functional role of NRF2 signaling in sphere cultured carcinoma cells

Abstract Cancer stem cells (CSCs) are known to express high levels of antioxidant proteins and ABC transporters. In this study, we investigated the potential involvement of NF-E2-related factor 2 (NRF2) in CSC survival and stress response by using the MCF7 mammosphere culture, which is accepted as a CSCs-enriched system. The levels of NRF2 protein, reporter transcription activity, and target gene expression such as efflux transporters were higher in mammospheres than in the monolayer. We observed that proteasome activity as well as catalytic subunits expression was decreased in the mammospheres. Additionally, mammospheres showed an elevated p62 protein level and p62-knockdown suppressed NRF2 activation. The role of NRF2 in mammospheres could be demonstrated in NRF2-knockdown mammospheres. Sphere growth was retarded in knockdown group and anticancer drugs resistance was not obtained in NRF2-knockdown mammospheres in contrast to the control mammospheres. NRF2 activation could be confirmed in spheres-culture of colon cancer and ovarian cancer cells. These results show that reduced proteasome function and elevated p62 level contributed to NRF2 activation in CSCs-enriched mammospheres. The study further suggests a potential role of NRF2 in CSCs survival and stress resistance. Citation Format: In-geun Ryoo, Mi-Kyoung Kwak. Functional role of NRF2 signaling in sphere cultured carcinoma cells. [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 2224. doi:10.1158/1538-7445.AM2015-2224

Quantitative Profiling of Lysine Acetylation Reveals Dynamic Crosstalk between Receptor Tyrosine Kinases and Lysine Acetylation.

Lysine acetylation has been primarily investigated in the context of transcriptional regulation, but a role for acetylation in mediating other cellular responses has emerged. Multiple studies have described global lysine acetylation profiles for particular biological states, but none to date have investigated the temporal dynamics regulating cellular response to perturbation. Reasoning that lysine acetylation may be altered in response to growth factors, we implemented quantitative mass spectrometry-based proteomics to investigate the temporal dynamics of lysine acetylation in response to growth factor stimulation in cultured carcinoma cell lines. We found that lysine acetylation changed rapidly in response to activation of several different receptor tyrosine kinases by their respective ligands. To uncover the effects of lysine acetylation dynamics on tyrosine phosphorylation signaling networks, cells were treated with an HDAC inhibitor. This short-term pharmacological inhibition of histone deacetylase activity modulated signaling networks involving phosphorylated tyrosine and thereby altered the response to receptor tyrosine kinase activation. This result highlights the interconnectivity of lysine acetylation and tyrosine phosphorylation signaling networks and suggests that HDAC inhibition may influence cellular responses by affecting both types of post-translational modifications.

Low-cost photodynamic therapy devices for global health settings: Characterization of battery-powered LED performance and smartphone imaging in 3D tumor models.

A lack of access to effective cancer therapeutics in resource-limited settings is implicated in global cancer health disparities between developed and developing countries. Photodynamic therapy (PDT) is a light-based treatment modality that has exhibited safety and efficacy in the clinic using wavelengths and irradiances achievable with light-emitting diodes (LEDs) operated on battery power. Here we assess low-cost enabling technology to extend the clinical benefit of PDT to regions with little or no access to electricity or medical infrastructure. We demonstrate the efficacy of a device based on a 635 nm high-output LED powered by three AA disposable alkaline batteries, to achieve strong cytotoxic response in monolayer and 3D cultures of A431 squamous carcinoma cells following photosensitization by administering aminolevulinic acid (ALA) to induce the accumulation of protoporphyrin IX (PpIX). Here we characterize challenges of battery-operated device performance, including battery drain and voltage stability specifically over relevant PDT dose parameters. Further motivated by the well-established capacity of PDT photosensitizers to serve as tumour-selective fluorescence contrast agents, we demonstrate the capability of a consumer smartphone with low-cost add-ons to measure concentration-dependent PpIX fluorescence. This study lays the groundwork for the on-going development of image-guided ALA-PDT treatment technologies for global health applications.

Sa1843 Cellular Localization and Function of Glypican3 (Gpc3) in Human Primary Culture of Hepatocellular Carcinoma (HCC) and Pre-HCC Cells

Sa1843 Cellular Localization and Function of Glypican3 (Gpc3) in Human Primary Culture of Hepatocellular Carcinoma (HCC) and Pre-HCC Cells

P6.08 - Cationic porphyrin-phenazine conjugates as quadruplex DNA ligands and potential agents for anticancer therapy

ABSTRACT Introduction: Telomeres are guanine-rich DNA sequences at the ends of chromosomes. Telomeric DNA forms G-quadruplexes (G4-DNA), and stabilization of these structures with small molecules can inhibit telomerase, an enzyme responsible for telomere elongation upon cellular division. It is highly active at majority of tumor cells, so G4-DNA and telomerase are considered as targets for the development of novel anticancer drugs. We present porphyrin conjugate with phenazine derivative and its metal complexes as selective G4-DNA ligands and potential antitumor agents. Materials and methods: Conjugates of tricationic porphyrin TMP3 and its Zn(II) and Mn(III) complexes with imidazo[4, 5-b]phenazine (ImPhz) intercalating agent were synthesized as previously reported [ 1 ]. Their binding affinity to G4 and dsDNA was studied using FID (Fluorescent Interacalator Displacement) method. The level of telomerase inhibition was determined by TRAP (Telomeric Repeat Amplification Protocol) in vitro assay. Antiproliferative activity of conjugates was tested on a mouse Lewis lung carcinoma cell culture. Results: All compounds show strong affinity to Tel22 G4-DNA. Dissociation constants for non-metalated conjugate, its Zn and Mn complex are 20.1, 46.3 and 3.8 µM, respectively. All ligands have a good selectivity for quadruplex over duplex DNA. The ratio between G4 and dsDNA constants is 5 for TMP3-ImPhz and 8 and 10 for its Zn and Mn complexes. High affinity of conjugates for G4-DNA is associated with a good ability to inhibit telomerase. The most efficient Zn complex completely inhibits the enzyme at 2.5 µM concentration being several times more active than non-conjugated porphyrins. Non-metalated conjugate shows inhibition effect at 5 µM, whereas Mn derivative is less active. Compounds display antiproliferative activity in vitro in micromolar range, with EC50 values for TMP3-ImPhz, its Mn and Zn complex to be 21.8, 11.2 and 5.9 µM, respectively. Conclusion: All studied compounds have strong binding affinity and selectivity for quadruplex DNA. They are efficient telomerase inhibitors and demonstrate good antiproliferative activity. Thus, these conjugates can be considered as potential agents for anticancer therapy.