Adenocarcinoma Cell Line Research Articles

17β-Estradiol Stimulates Generation of Reactive Species Oxygen and Nitric Oxide in Ovarian Adenocarcinoma Cells (OVCAR 3).

Background:Experimental and epidemiological evidence supports a role for steroid hormones in the pathogenesis of ovarian cancer. Among steroid hormones, 17β-estradiol (E2) has the most potent effect on proliferation, apoptosis and metastasis. Objectives:In the present study, we investigated the effect of E2 on production of ROS and NO in ovarian cancer cells.Materials and Methods:Ovarian adenocarcinoma cell line (OVCAR-3) was cultured and treated with various concentrations of E2, antioxidants (N-acetyle cysteine and Ebselen) and ICI182780 as an estrogen receptor antagonist. MTT test was performed to evaluate cell viability. NO and ROS levels were measured by Griess and DCFH-DA methods, respectively.Results:ROS levels as well as NO levels were increased in OVCAR-3 cells treated with E2. The increase in ROS production was in parallel with increased cell viability which indicates that estrogen-induced ROS can participate in cancer progression. ICI182780 abolished E2-induced ROS production. Progesterone was also effective in reducing ROS and NO generation. Conclusions:NO and ROS are important molecules in signaling networks in cell. These molecules can be used as therapeutic targets for prevention and treatment of ovary cancer and other estrogen-induced malignancies.

New benzimidazole-derived isothioureas as potential antileukemic agents--studies in vitro.

Isothioureas are a class of amphiphilic compounds carrying a highly basic isothiourea group of pKa ranging between 10 and 11. Hence, they exist in protonated (cation) form at physiological pH, a characteristic is of key importance for their pharmacological activity. Recently, we have found that a number of S-pentabromobenzylisothiourea derivatives show substantial cytotoxicity toward a variety of human glioblastoma, leukemia, and adenocarcinoma cell lines. Whereas there is a growing body of data on aliphatic and alkylaromatic isothioureas, little attention was given to this day to heterocyclic isotiourea derivatives. Here we report on the synthesis and pharmacological in vitro properties of 10 novel S-(benzimidazol-2-ylmethyl)- and S-(5,6-dichlorobenzimidazol-2-ylmethyl)isothiourea derivatives. The compounds were obtained by the condensation of the respective 2-chloromethyl benzimidazoles with various substituted N(N')-thioureas. Besides the essential physicochemical characteristics (H-NMR, UV, elemental analysis) of the new compounds, their log P values, which are of key importance for in vivo drug distribution and interactions, were determined. Pharmacological (anticancer) activity of the compounds was evaluated based on their ability to induce apoptosis in exponentially growing cultures of the human acute myelogenous leukemia cell line KG-1; the apoptosis was assessed with a variety of flow cytometric methods. Of the novel compounds tested, the most potent apoptosis inducer in KG-1 cells was N-methyl-S- (5,6-dichloro-1H-benzimidazol-2-ylmethyl)isothiouronium chloride (ClBMMe).

SOX4 interacts with EZH2 and HDAC3 to suppress microRNA-31 in invasive esophageal cancer cells.

BackgroundTumor metastasis is responsible for 90% of cancer-related deaths. Recently, a strong link between microRNA dysregulation and human cancers has been established. However, the molecular mechanisms through which microRNAs regulate metastasis and cancer progression remain unclear.MethodsWe analyzed the reciprocal expression regulation of miR-31 and SOX4 in esophageal squamous and adenocarcinoma cell lines by qRT-PCR and Western blotting using overexpression and shRNA knock-down approaches. Furthermore, methylation studies were used to assess epigenetic regulation of expression. Functionally, we determined the cellular consequences using migration and invasion assays, as well as proliferation assays. Immunoprecipitation and ChIP were used to identify complex formation of SOX4 and co-repressor components.ResultsHere, we report that SOX4 promotes esophageal tumor cell proliferation and invasion by silencing miR-31 via activation and stabilization of a co-repressor complex with EZH2 and HDAC3. We demonstrate that miR-31 is significantly decreased in invasive esophageal cancer cells, while upregulation of miR-31 inhibits growth, migration and invasion of esophageal adenocarcinoma (EAC) and squamous cell carcinoma (ESCC) cell lines. miR-31, in turn, targets SOX4 for degradation by directly binding to its 3′-UTR. Additionally, miR-31 regulates EZH2 and HDAC3 indirectly. SOX4, EZH2 and HDAC3 levels inversely correlate with miR-31 expression in ESCC cell lines. Ectopic expression of miR-31 in ESCC and EAC cell lines leads to down regulation of SOX4, EZH2 and HDAC3. Conversely, pharmacologic and genetic inhibition of SOX4 and EZH2 restore miR-31 expression. We show that SOX4, EZH2 and HDAC3 form a co-repressor complex that binds to the miR-31 promoter, repressing miR-31 through an epigenetic mark by H3K27me3 and by histone acetylation. Clinically, when compared to normal adjacent tissues, esophageal tumor samples show upregulation of SOX4, EZH2, and HDAC3, and EZH2 expression is significantly increased in metastatic ESCC tissues.ConclusionsThus, we identified a novel molecular mechanism by which the SOX4, EZH2 and miR-31 circuit promotes tumor progression and potential therapeutic targets for invasive esophageal carcinomas.

Elevated circulating tissue inhibitor of metalloproteinase 1 (TIMP-1) levels are associated with neuroendocrine differentiation in castration resistant prostate cancer.

Tissue inhibitor of metalloproteinase-1 (TIMP-1) is a 28.5 kDa secreted glycoprotein that inhibits matrix metalloproteinase (MMP) activity. Our group has previously shown that elevated plasma TIMP-1 levels predict poor survival in metastatic castration-resistant prostate cancer (CRPC) patients; however, the underlying source and impact of elevated circulating TIMP-1 protein is unknown. In this study, we used qRT-PCR, ELISA and immunohistochemistry to evaluate TIMP-1 expression in androgen-sensitive and resistant prostate cancer (PC) cell lines, tumor tissues and patient sera, and to correlate TIMP-1 levels to expression of chromogranin A (CGA), an established marker of neuroendocrine differentiation (NED). We also explored the relationship between TIMP-1 overexpression and induction of NED by overexpressing TIMP-1 in androgen-sensitive LNCaP cells, as well as by inducing NED of LNCaP cells with IL-6. Patients with CRPC have significantly higher serum TIMP-1 levels compared to patients with hormone-sensitive disease. Although circulating TIMP-1 levels were increased, peripheral blood cells were not the source of elevation. Instead, elevated TIMP-1 expression was associated with higher expression of CGA in both blood and metastatic tumor tissue. We further show that androgen receptor (AR) and PSA non-expressing prostate cancer cell lines known to display NED phenotypes such as PC-3, PC-3M, and DU145 cells, expressed high levels of TIMP-1, in contrast to AR (+) and PSA (+) adenocarcinoma cell lines such as LNCaP, VCaP, and LAPC-4, which had barely detectable levels of TIMP-1. In addition, ectopic overexpression of TIMP-1 in LNCaP cells did not induce NED. However, TIMP-1 mRNA expression was elevated >10-fold during IL-6-induced NED of LNCaP cells, suggesting that TIMP-1 overexpression accompanies, but is not the driving force for NED. Finally, we show that conditioned media from androgen-resistant PC-3, PC-3M, and DU145 cells induced TIMP-1 mRNA expression in primary prostate stromal fibroblasts in an ERK and NF-κB dependent manner. We provide in vitro and clinical evidence to support the association between NED and elevated circulating TIMP-1 expression in CRPC. Our observation supports further evaluation of TIMP-1 as a tissue and serum biomarker for NED in CRPC.

Isolation of Flavonoids and their Anticancer Activity fromSphaeranthus amaranthoidesin A549 Cell Line

Flavonoids are considered as potential explore to treat cancer and they are ubiquitous. The chemical structure and the nature of the polyphenolic flavonoids are responsible for their anticancer actions. Objective: The present investigation concentrates on the cytotoxic effect of flavonoids in A549 adenocarcinoma cell lines. Methodology: the plant extract was subjected to TLC for confirmation and HPLC confirm the pure form of the flavonoids and the induction of the cytotoxicity was studied using AO/EB, PI and the uptake of the PI was confirmed using the FACS in A549 cells. results: the TLC and HPLC results confirmed the presence of flavonoides. Flow cytometry denotes the percentage of the cells in the G0/G1 and the G2 stage. This indicates the accumulation of the cells in the apoptotic and proapoptotic stages. Conclusion: These findings indicate that the flavonoids from the Sphaeranthus amarathoides may be promising anticancer agents to control the cell growth in A549 cells.

Ursane Saponins from the Stems ofFirmiana simplexand their Cytotoxic Activity

Three new ursane triterpene saponins, together with twelve known ursane triterpenes were isolated from the stems of Firmiana simplex. The structures of the saponines were elucidated on the basis of spectroscopic and chemical methods. The cytotoxic activity of all compounds was evaluated in vitro against lung adenocarcinoma (A549), ovarian cancer (SK-OV-3), skin melanoma (SK-MEL-2), and colon cancer (HCT-15) human cell lines, using a sulforhodamine (SRB) assay. 23-Hydroxyursolic acid showed cytotoxicity against the tested cell lines with IC50 values ranging from 11.96 to 14.11 µM.

Bucky Tubes Induce Oxidative Stress Mediated Cell Death in Human Lung Cells.

Unique physicochemical properties of carbon nanomaterials (CNMs) have opened a new era for therapeutics and diagnosis (known as theranostics) of various diseases. This exponential increase in application makes them important for toxicology studies. The present study was aimed at exploring the toxic potential of one of the CNMs, that is, bucky tubes (BTs), in human lung adenocarcinoma (A549) cell line. BTs were characterised by electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform spectroscopy (FTIR), and X-ray diffraction (XRD). Flow cytometric study showed a concentration and time dependent increase in intracellular internalization as well as reduction in cell viability upon exposure to BTs. However, a significant increase in intracellular reactive oxygen species (ROS) production was observed as evident by increased fluorescence intensity of 2′,7′-dichlorofluorescein (DCF). BTs induced oxidative stress in cells as evident by depletion in glutathione with concomitant increase in lipid peroxidation with increasing concentrations. A significant increase in micronucleus formation and apoptotic cell population and loss of mitochondrial membrane potential (MMP) as compared to control were observed. Moreover, in the present study, BTs were found to be mild toxic and it is encouraging to conclude that BTs having outer diameter in the range of 7–12 nm and length 0.5–10 μm can be used for theranostics.

Next-generation sequencing reveals low-dose effects of cationic dendrimers in primary human bronchial epithelial cells.

Gene expression profiling has developed rapidly in recent years with the advent of deep sequencing technologies such as RNA sequencing (RNA Seq) and could be harnessed to predict and define mechanisms of toxicity of chemicals and nanomaterials. However, the full potential of these technologies in (nano)toxicology is yet to be realized. Here, we show that systems biology approaches can uncover mechanisms underlying cellular responses to nanomaterials. Using RNA Seq and computational approaches, we found that cationic poly(amidoamine) dendrimers (PAMAM-NH2) are capable of triggering down-regulation of cell-cycle-related genes in primary human bronchial epithelial cells at doses that do not elicit acute cytotoxicity, as demonstrated using conventional cell viability assays, while gene transcription was not affected by neutral PAMAM-OH dendrimers. The PAMAMs were internalized in an active manner by lung cells and localized mainly in lysosomes; amine-terminated dendrimers were internalized more efficiently when compared to the hydroxyl-terminated dendrimers. Upstream regulator analysis implicated NF-κB as a putative transcriptional regulator, and subsequent cell-based assays confirmed that PAMAM-NH2 caused NF-κB-dependent cell cycle arrest. However, PAMAM-NH2 did not affect cell cycle progression in the human A549 adenocarcinoma cell line. These results demonstrate the feasibility of applying systems biology approaches to predict cellular responses to nanomaterials and highlight the importance of using relevant (primary) cell models.

Abstract B47: Therapeutic KRAS silencing in lung and colon cancer models

Abstract Introduction: Despite being amongst the most common oncogenes in human cancer, to date there are no effective clinical options for inhibiting KRAS activity. We investigated whether systemically delivered KRAS siRNAs using biocompatible nanoparticles has therapeutic potential in KRAS mutated lung and colorectal cancer models. Methods: We identified two KRAS siRNA sequences with notable potency in knocking-down KRAS expression independent of nucleotide substitution. Using human lung (A549; KRASG12S) and colon (HCP1; KRASG12D) adenocarcinoma cell lines, we assessed anti-proliferative effects of KRAS silencing with MTT assays in vitro. For in vivo experiments, we used a nano-liposomal delivery platform, DOPC, for systemic delivery of siRNAs. We used luciferase-labeled A549 cells to create an orthotopic lung cancer model in athymic nude mice. One week after cell injection, we began weekly IVIS imaging and treatments to assess efficacy of systemic KRAS siRNA with and without cisplatin (CDDP) chemotherapy. Proliferation and apoptosis indices were assessed using Ki-67 and caspase-3 IHC, respectively. Using a patient-derived xenograft colon cancer cell line, HCP1, we performed several in vivo experiments to assess KRAS siRNA effects on down-stream signaling, primary tumor growth, and experimental metastasis. Results: For both KRAS siRNA sequences, greater than 90% knock-down of KRAS was observed by western blot; ∼40-60% reductions in cell viability was observed in both A549 and HCP1 (p<0.001). In the lung cancer model, following four weeks of systemic therapy, the KRAS siRNA treatment group demonstrated an 81% reduction in luminescence compared to mice in the NC siRNA group (p=0.008). In addition, only in the KRAS siRNA treatment groups did we observe significant reductions in primary tumor size (KRAS siR: 50%, p=0.003; NC siR+CDDP: 6%, p=0.24; KRAS siR+CDDP: 73%, p=0.008), number of distant metastases (KRAS siR: 71%, p=0.07; NC siR+CDDP: 0%, p=0.49; KRAS siR+CDDP: 81%, p=0.02) and aggregate mass of distant metastases (KRAS siR: 77%, p=0.0002; NC siR+CDDP: 11%, p=0.85; KRAS siR+CDDP: 74%, p=0.009). KRAS siRNA significantly reduced the frequency of mediastinal metastases (NC siR: 100% versus KRAS siR: 25%, p=0.002). Compared with control siRNA, significant reductions in Ki-67 indices were seen in all treatment groups (KRAS siR: 34%, NC siR+CDDP: 52%, KRAS siR+CDDP: 36%; all p<0.0001), while significant increases in caspase-3 activity was only seen in the CDDP treatment groups (NC siR+CDDP: 2-fold increase, p=0.03; KRAS siR+CDDP: 5.5-fold increase, p=0.0007). Using the HCP1 colon cancer model, KRAS siRNA treatment reduced tumor KRAS and pERK expression levels at 48 and 96 hours after systemic delivery. Consistent with these effects, as compared with NC siRNA, treatment with KRAS siRNAs reduced HCP1 subcutaneous tumor growth by 69% (p<0.05). To assess effects of KRAS siRNA on liver metastases, HCP1 cells were delivered by intra-splenic injection followed by splenectomy the following day. Following 3 weeks of treatment with KRAS siRNAs, we observed reduced aggregate liver metastatic tumor burden compared to controls (73%, p=0.014). Notably, no mice in any of the therapeutic experiments exhibited any overt signs of toxicity from KRAS siRNA treatment, such as decreased feeding habits, activity, or total body weight. Discussion: Our studies demonstrate a proof-of-concept approach to therapeutic KRAS targeting using nanoparticle delivery of siRNA. This study highlights the potential translational impact of therapeutic RNA interference, which may have broad applications in oncology, especially for traditional “undruggable” targets. Citation Format: Chad Pecot, Sherry Wu, Seth Bellister, Rajat Bhattacharya, Anshumaan Maharaj, Cristian Rodriguez-Aguayo, Vianey Gonzalez-Villasana, Rajesha Rupaimoole, Gabriel Lopez-Berestein, Lee M. Ellis, Anil Sood. Therapeutic KRAS silencing in lung and colon cancer models. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B47. doi: 10.1158/1557-3125.RASONC14-B47

Establishment of a human lung adenocarcinoma A549 cell line with RNA interference for Y-box binding protein-1 gene silencing and the influence on A549 cell proliferation

Objective To establish a human lung adenocarcinoma A549 cell line with RNA interference for YB-1 gene silencing which can stably express green fluorescent protein (GFP) and to observe the effects of silencing Y-box binding protein (YB-1) gene on proliferation in lung adenocarcinoma A549 cells.Methods The human lung adenocarcinoma A549 cells were transfected with shRNA plasmid specific for the YB-1 gene,and were then selected through G418.G418-resistant clones were generated and screened using reverse transcription polymerase chain reaction (RT-PCR),real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) and Western blotting analysis.Cell proliferation was examined by methyl thiazolyl tetrazolium.Results The G418-resistant clones (A549-746 and A549-326) showed much lower level of YB-1 mRNA (0.41 ± 0.01 and 0.39 ± 0.06) than A549-shNC (P ＜ 0.05) under RT-PCR.A549-746 and A549-326 cell clones showed much lower level of YB-1 mRNA (0.43 ±0.10 and 0.20 ± 0.24) and protein expression (0.49 ± 0.06 and 0.24 ± 0.02) than A549-shNC (P ＜ 0.05) under FQ-PCR and Western blotting analysis.Meanwhile,the G418-resistan clones (A549-746 and A549-326) stably expressed GFP in vitro.The cell growth rate in YBX1-homo-746 and YBX1-homo-326 groups was decreased significantly at the first 24 h (P ＜ 0.05).Conclusion The lung cancer cell lines A549-746 and A549-326 with stable YB-1 gene silencing and GFP expression were successfully established,and silencing YB-1 gene could inhibit the proliferation of lung adenocarcinoma A549 cells. Key words: Lung adenocarcinoma; Y-box binding protein-1 ; Short hairpin RNA; Green fluorescent protein

Apoptotic induction of lung adenocarcinoma A549 cells infected by recombinant RVG Newcastle disease virus (rL-RVG) in vitro.

Newcastle disease virus (NDV) is a member of the genus Avulavirus in the Paramyxoviridae family and its antitumor properties depend on its ability to kill malignant cells while not affecting normal cells. The present study investigated a recombinant avirulent NDV LaSota strain (wild-type NDV strain) expressing the rabies virus glycoprotein (rL-RVG), examined its oncolytic effect on the lung adenocarcinoma A549 cell line and evaluated its potential to serve as a vaccine against lung cancer. A549 cells were infected with the rL-RVG virus and analyzed by MTT, western blot, polymerase chain reaction (PCR), immunofluorescence, terminal deoxynucleotidyl transferase dUTP nick end labeling and flow-cytometric analyses. PCR, western blot and immunofluorescence showed that the RVG gene and protein were stably expressed in A549 cells following infection with rL-RVG. The growth of A549 cells in the rL-RVG group was inhibited more effectively compared to those infected with the wild-type NDV strain. MTT results showed that cell growth inhibition rates in the rL-RVG group were significantly higher than those in the NDV group (P<0.05). Early apoptosis in the rL-RVG group was also more evident, with the apoptotic index being increased in rL-RVG group. The expression of the pro-apoptotic proteins caspase-3, -8 and -9 increased. The expression of caspase-3 decreased following application of the broad-specificity caspase inhibitor Z-VAD-FMK. However, the expression of the inhibitory apoptosis protein B-cell lymphoma 2 (bcl-2) did not change, but bcl-2-associated X/bcl-2 ratio was higher in the rL-RVG group than that in the NDV group. The rL-RVG strain was able to suppress lung cancer cell growth and promote lung cancer cell apoptosis to a greater extent than the wild-type NDV strain. Therefore, the rL-RVG strain is a potent antitumor agent.

Abstract 391: Identification of a novel tumor-suppressor gene through methylome analysis in smoking-associated lung adenocarcinoma

Abstract Aberrant methylation of promoter DNA and transcriptional repression of specific tumor suppressor genes (TSGs) play an important role in carcinogenesis. In order to identify novel TSG candidates implicated in smoking-associated lung adenocarcinoma (AC), we performed genome-wide methylation profiling of six smoking-associated lung ACs with paired non-cancerous tissues using the single-CpG resolution Infinium array. After defining differentially methylated candidates including known targets in ACs, we analyzed gene expression using microarray/RT-PCR to identify genes potentially silenced by promoter methylation. Frequent hypermethylation and downregulation of candidates was further validated by pyrosequencing and RT-PCR/immunohistochemistry, respectively, in a larger set of ACs, and methylation dependency of silencing was determined by 5-aza-2-deoxycytidine treatment and promoter assay in AC cell lines. Through this approach, we identified one gene matching all of our selection criteria for a possible TSG (HSLAC1, Lab. name). Additionally, functional analyses supported a tumor-suppressive activity in vitro. Our results implicate HSLAC1 as a novel TSG for smoking-associated lung AC. Citation Format: Issei Imoto. Identification of a novel tumor-suppressor gene through methylome analysis in smoking-associated lung adenocarcinoma. [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 391. doi:10.1158/1538-7445.AM2014-391

Abstract 4062: Nestin regulates proliferation, migration, invasion and stemness of lung adenocarcinoma

Abstract Lung cancer is the most common cancer and the most common cause of cancer-related death in the world. Despite of the therapeutic advances in the treatment of non-small cell lung cancer (NSCLC) including molecular targeted therapy for lung adenocarcinoma (AD), the prognosis of the patients with NSCLC is dismal. Therefore, new treatment strategies are needed. Nestin, a class VI intermediate filament, is known to be a cancer stem cell (CSC) marker as well as a neuroepithelial stem cell marker. High expression levels of nestin are reported in various malignant tumors including lung, pancreas, cervical and prostate cancers, malignant melanomas and glioblastomas. Nestin is thought to regulate tumor cell proliferation, migration, invasion and CSC properties. Here we confirmed nestin expression in NSCLC: Immunohistochemical analysis in surgical specimens detected nestin protein expression in the cytoplasm of 20 of 48 AD cases (41.7%) and 25 of 47 squamous cell carcinoma cases (53.2%). Nestin immunoreactivity significantly correlated with not only tumor size and lymph node metastasis in NSCLC (p = 0.02, for both), but also poor survival in surgical patients with AD (p = 0.02). High and moderate expression levels of nestin were confirmed in several lung AD cell lines including H1975 and PC-3. Nestin inhibition by short hairpin RNA (shRNA) decreased proliferation, migration, invasion, and sphere formation in AD cell lines. Correspondingly, nestin upregulation by nestin gene transfection in these cells resulted in the opposite changes. Moreover, we performed screening analysis to clarify the upstream molecule that regulates nestin expression, using 95 chemical compounds that each inhibited a specific molecular target. Akt inhibitor IV led to a 72% reduction of nestin expression compared to in the DMSO-treated negative control in PC-3 cells. Akt is located downstream of the EGFR pathway, and Akt is reported to regulate SRY-box containing protein 2 (Sox2) expression and the self-renewal of CSC-like cells in NSCLC. Sox2 is also known to be a transcriptional factor for nestin. Immunohistochemically, we also confirmed co-expression of nestin, Akt, phosphorylated Akt, and Sox2 in serial tissue sections from AD cases. Thus, we performed further experiments using Akt inhibitor IV to clarify the relationship between Akt, Sox2, and nestin in AD cells. Akt inhibitor IV effectively decreased nestin expression via Sox2 downregulation and overcame the enhanced sphere formation induced by nestin upregulation. These findings suggest that nestin correlates to the aggressiveness and stemness of AD. Inhibition of nestin via Akt/Sox2 is thus a novel therapeutic approaches to eradicate lung AD and CSCs. Citation Format: Kosuke Narita, Yoko Matsuda, Masahiro Seike, Zenya Naito, Akihiko Gemma, Toshiyuki Ishiwata. Nestin regulates proliferation, migration, invasion and stemness of lung adenocarcinoma. [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 4062. doi:10.1158/1538-7445.AM2014-4062

Abstract 2276: Induction of apoptosis and inhibition of angiogenesis by novel fusion protein - AD-O54.9 as a new preclinical strategy in cancer treatment

Abstract Background For almost two decades tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) has been under extensive development as a potential therapeutic agent. TRAIL/Apo2L is a member of the TNF superfamily with unique ability to induce apoptosis in cancer cells while remaining neutral to normal cells. Tumor growth is tightly related to new blood vessel formation and tissue remodeling. Platelet derived growth factor (PDGF) is important in vascular physiological and pathological development. Inhibition of PDGF pathway blocks angiogenesis what results in reduction of tumor growth. We proposed a novel fusion protein (AD-O54.9) with dualistic proapoptotic and antiangiogenic activity. Our molecule consists of the soluble recombinant TRAIL/Apo2L variant linked with an effector peptide sequence and an activation motif recognized by tumor-specific proteases (MMPs, uPa) between. The effector peptide is composed of 19-amino acid fragment derived from human PDGF, which binds the PDGF receptors competitively to the natural ligand while being itself devoid of activity. As a consequence, angiogenic activity of PDGF is blocked, stimulation of new blood vessels formation does not occur and finally tumor growth is inhibited. Materials and methods AD-O54.9 protein was expressed in E. coli, using pET expression system, and purified by IEC chromatography. Cytotoxic activity of AD-O54.9 was examined using a propidium iodide assay and its antiangiogenic activity was evaluated by HUVEC tube formation and ring aortic assays. Safety was tested on human primary hepatocytes. The proapoptotic and antiproliferative activity was tested using molecular biology and flow cytometry methods. In vivo potential was examined on mice xenograft models of human colorectal adenocarcinoma (Colo205), human lung carcinoma (NCI-H460-luc2), human pancreatic carcinoma (MIA PaCa-2) and human oesophageal adenocarcinoma (OE19) cell lines where molecules showed anticancer activity. Results The molecule showed in vitro specific cytotoxic effect on various primary cancer cell lines at IC50 below 0.01 ng/ml. We demonstrated that AD-O54.9 is a very potent apoptosis inducer and inhibitor of angiogenesis. This fusion protein showed superior efficacy displaying significant tumor volume regression compared with TRAIL/Apo2L and standard chemotherapeutic agents. Conclusions The obtained results confirm that we developed a very promising fusion molecule with a high potential of anticancer activity that could be considered as a novel therapeutic agent. Citation Format: Piotr Rózga, Jerzy Pieczykolan, Bartłomiej Zerek, Anna Pieczykolan, Marlena Gałązka, Katarzyna Bukato, Michał Szymanik, Albert Jaworski, Sebastian Pawlak, Małgorzata Teska-Kamińska, Anna Grochot-Przęczek. Induction of apoptosis and inhibition of angiogenesis by novel fusion protein - AD-O54.9 as a new preclinical strategy in cancer treatment. [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 2276. doi:10.1158/1538-7445.AM2014-2276

Abstract 4497: Therapeutic benefit of targeting ERK in mutant KRAS pancreatic ductal adenocarcinoma

Abstract The limited success of anti-MEK and anti-RAF therapies with KRAS mutant cancers has been attributed, in part, to de novo or acquired resistance mechanisms that arise after prolonged treatment. A majority of resistance mechanisms involve kinome reprograming to reactivate ERK downstream of inhibitor activity. Therefore, we hypothesized that directly targeting ERK would be an effective method for overcoming these resistance mechanisms. To address this possibility, we evaluated the sensitivity of a panel of mutant KRAS pancreatic ductal adenocarcinoma (PDAC) cell lines to the highly selective SCH772984 ERK1/2 protein kinase inhibitor. First, we found that the anchorage-dependent growth of 6 of 12 cell lines were sensitive to SCH772984 treatment (IC50&amp;lt;2 uM). Some sensitive PDAC lines were resistant to the AZD6244 MEK1/2-selective inhibitor, indicating different mechanisms of action of these two inhibitors. Second, we identified phospho-AKT as a potential biomarker for PDAC SCH772984 sensitivity. Third, we assessed the molecular basis for anchorage-dependent growth inhibition. After 72 h treatment, flow cytometry analyses indicated impaired cell cycle progression through G1. Additionally, we found increased apoptosis as measured by formation of cleaved caspase-3. Together these observations suggest that sensitivity is a result of both impaired cell cycle progression and induction of apoptosis. Surprisingly prolonged SCH772984 treatment (2 weeks) resulted in senescence, as sensitive lines stained positive for senescence-associated beta-galactocidase. Furthermore, we found that senescent cells induced p16 and lost phospho-RB two common markers associated with induction of senescence. Onset of senescence was dependent on the loss of total MYC protein expression. Finally, we found that treatment with SCH772984 reduced tumorigenic growth in an in vivo xenograft mouse model. Our ongoing studies include determining the mechanisms of inhibitor resistance. In summary, our findings suggest that targeting ERK will be therapeutically beneficial for the treatment of KRAS mutant PDAC. Citation Format: Tikvah K. Hayes. Therapeutic benefit of targeting ERK in mutant KRAS pancreatic ductal adenocarcinoma. [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 4497. doi:10.1158/1538-7445.AM2014-4497

Abstract 1977: Therapeutic miRNAs targeted selectively to tumors by mesenchymal stem cell derived microparticles

Abstract Introduction: MiRNAs have been implicated in the development of some if not all cancer types and have been identified as attractive targets for therapy. However, systemic delivery of bare miRNAs faces its own set of limitations because of degradation by RNases and filtration and excretion by the kidneys. In this work we propose miRNAs as therapeutic agents. We have developed a novel system for miRNA delivery applicable for both local and systemic administration with the use of mesenchymal stem cell (MSC) microparticles (MPs). Methodology: MSCs were isolated from the Wharton's jelly of human umbilical cords. The MSC cultures were subjected to serum deprivation, leading to the formation of MPs (secreted membrane vehicles &amp;lt;1μm) which were harvested and characterized by SEM, PCR, FACS and Fluorescence Microscopy. Breast (MDA-MB-231 &amp; MCF-7), colon (RKO &amp; HT-29) and ovarian adenocarcinoma (SKOV3) cell lines were then exposed to MPs. The response to treatment was evaluated by cell morphology, proliferation, migration, gene expression and apoptosis. Furthermore, the therapeutic potential of MPs was tested in vivo in xenograft tumor mouse models. Innovative imaging modalities such as in vivo flow cytometry and whole body fluorescence-bioluminescence were employed to dynamically investigate the biodistribution and homing kinetics of MPs in mice. Results: In vitro experiments confirmed that MSC-derived MPs can be internalized by the various cancer cell lines and induce a biological effect as evidenced by membrane damage, cell shrinkage and blebbing in the recipient cell. Significantly, there was evidence that MPs induce apoptosis, inhibit cell proliferation and tumor growth attenuation in a dose/time-dependent manner. The pro-apoptotic and anti-migration effects of MPs in cancer cells were almost completely abrogated by RNase treatment before administration to cultures. In vivo studies demonstrated that we were able to monitor and quantify fluorescently labelled MPs in circulation and to detect and image the biodistribution and incorporation in cells and organs in healthy and tumor-bearing mice. Conclusion: MSC-derived MPs containing miRNAs possess tumor inhibitory properties both in vitro and in vivo. Administration of MPs after RNase treatment induces the loss of anti-cancer properties suggesting a horizontal transfer of small RNAs from MPs to cancer cells. MPs formulated to contain specific miRNAs, could affect the action of genes associated with carcinogenesis, neovascularization, metastasis and other cancer characteristics, thus leading to therapeutic benefit. Citation Format: Marianna Prokopi, Agamemnon Epenetos, Andreas Anayiotos, Costas Pitsillides, Konstantinos Kapnisis, Christina Kousparou. Therapeutic miRNAs targeted selectively to tumors by mesenchymal stem cell derived microparticles. [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 1977. doi:10.1158/1538-7445.AM2014-1977

Abstract 1420: RRx-001 inhibits glucose erythrocyte and tumor glucose 6-phosphate dehydrogenase

Abstract Cancer cells are characterized by an excess of reactive oxygen species (ROS) levels, which are balanced by high levels of glutathione (GSH) to protect against ROS-induced cytotoxicity. Glucose 6-phosphate dehydrogenase (G6PD), the first key enzyme of the pentose phosphate pathway (PPP), ensures NADPH supply for reduced glutathione regeneration. RRx-001, a novel redox active anticancer agent, derived from the aerospace industry, which has successfully completed a Phase 1 clinical trial and is scheduled to begin multiple Phase 2 studies, induces cell cycle-specific apoptosis through increase in intracellular ROS leading to free thiol depletion. The purpose of the present study was to evaluate whether the mechanism by which RRx-001 targets the antioxidant capacity of tumor cells is related to G6PD inhibition. Since red blood cells (RBC) are particularly dependent on G6PD synthesis, we initially explored the effects of RRx-001 on the erythrocytic PPP. Glucose consumption was higher for human RBC (hRBC) treated with RRx-001 (10 μM) compared to untreated cells. PPP was inhibited in RRx-001 treated hRBC. Methylene blue, which normally stimulates PPP flux, did not increase glucose utilization of treated cells. Analysis of PPP flux revealed that RRx-001 inhibits G6PD preventing conversion of glucose-6-phosphate to 6-phosphogluconolactone and reduction of NADP(+). Compared to G6PD inhibitors dehydroepiandrosterone and aminonicotinamide, RRx-001 was 10 fold more potent. Given the critical importance of G6PD and NADPH to fuel both the proliferation of tumor cells and to detoxify ROS, we secondarily investigated whether RRx-001 has anti-proliferative and viability effects on hepatocellular carcinoma (Hep G2) and colorectal adenocarcinoma (Caco-2 and HT-29) cell lines. Through tetrazolium and bromodeoxyuridine incorporation assays, performed as indicators of cellular proliferation, RRx-001 inhibited cell growth in a dose dependent (1 - 20 μM) manner. The suppression of growth of the RRx-001 incubated cells was correlated with inhibition of G6PD (r=0.81, n=9, P&amp;lt;0.05). RRx-001 also reduced viability of all carcinoma cell lines. Given the key roles of the PPP in GSH metabolism, and the observation that cell lines overexpressing G6PD are associated with the formation of tumors in mice, inhibition of G6PD by RRx-001 is an attractive target, particularly in combination with standard of care radiotherapy or cytotoxic drugs that also generate ROS or free radical intermediates either directly or indirectly. Citation Format: Pedro Cabrales, Bryan Oronsky, Jan Scicinski. RRx-001 inhibits glucose erythrocyte and tumor glucose 6-phosphate dehydrogenase. [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 1420. doi:10.1158/1538-7445.AM2014-1420

Abstract 1541: Co-mutations define major subsets of KRAS-driven lung adenocarcinoma with implications for targeted cancer therapy

Abstract Introduction The molecular underpinnings that beget the considerable biological and clinical heterogeneity of lung adenocarcinoma (AC) driven by oncogenic KRAS are poorly characterized. We hypothesized that significantly co-occurring mutations in components of key cellular tumor suppressor pathways are major determinants of signalling diversification downstream of mutant KRAS. Methods We applied un-supervised hierarchical analysis and classical multi-dimensional scaling to determine patterns of somatic (co)-mutations within a curated set of significantly mutated genes (MutSigCV p value &amp;lt;0.001) among 99 KRAS-mutant lung ACs from the TCGA dataset. Independently, we derived expression clusters by applying un-supervised hierarchical analysis to a subset of 68 KRAS-driven lung ACs with available RNASeq data. Non-negative matrix factorization (NMF) was used to determine cluster stability. Results Clustering of KRAS-driven lung ACs based on patterns of somatic mutations revealed six major mutation clades. Five clades could be defined by combinations of somatic mutations in only four genes : KRAS, TP53, STK11/LKB1 and KEAP1, whereas a sixth clade appeared more heterogenous and was associated with a higher overall rate of somatic mutation. Co-mutations in STK11/LKB1 and TP53 accounted for higher branches of the cluster dendrogram and were largely non-overlapping in the context of early-stage disease. Integration of co-mutation analysis with unsupervised hierarchical analysis of RNA Seq differential expression data identified 3 major expression clusters, that were significantly enriched for the KRAS;TP53 (KP), KRAS;LKB1 (KL) and KRAS only (K) somatic mutation clades, as well as sub-clusters characterized by the presence of mutations in KEAP1. On the contrary, the distribution of the main KRAS amino acid substitutions (G12C, G12V and G12D) did not differ significantly between the expression clusters, indicating that co-mutations rather than distinct KRAS codon 12 base substitutions are the major driver of biological heterogeneity within KRAS-mutant lung ACs. Re-analysis of publicly available large scale drug sensitivity data based on the proposed sub-classification revealed novel genotype-specific therapeutic vulnerabilities for the KL and KP subgroups including selective sensitivity of KL AC cell lines to MK-2206 ( a pan-AKT inhibitor), BX-795 (a PDK1 and TBK1/IKKϵ inhibitor) and the type II topoisomerase inhibitor etoposide. Selected hits were subsequently validated using pairs of isogenic cell lines. Conclusions Co-mutations in a small number of key tumor suppressor genes define biologically distinct and therapeutically relevant subgroups of KRAS-driven lung AC. Efforts to target KRAS signalling in the context of specific co-mutations may provide a more rational and personalized approach to therapy for this challenging group of lung cancer patients. Citation Format: Ferdinandos Skoulidis, Kevin R. Coombes, Lixia Diao, Pan Tong, Maria A. Cortez, Uma Giri, Chao Yang, You Hong Fan, John N. Weinstein, Vassiliki Papadimitrakopoulou, John D. Minna, Jing Wang, Lauren A. Byers, John V. Heymach. Co-mutations define major subsets of KRAS-driven lung adenocarcinoma with implications for targeted cancer therapy. [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 1541. doi:10.1158/1538-7445.AM2014-1541

CCR9/CCL25 expression in non-small cell lung cancer correlates with aggressive disease and mediates key steps of metastasis.

Poor clinical outcome of lung cancer (LuCa) is primarily due to lack of knowledge about specific molecules involved in its progression and metastasis. In this study, we for the first time show the clinical and biological significance of CC chemokine receptor-9 (CCR9) in non-small cell lung cancer (NSCLC). Expression of CCR9 and CCL25, the only natural ligand of CCR9, was significantly higher (p<0.0001) in NSCLC tissues and serum respectively, compared to their respective controls. Interestingly, expression of both CCR9 and CCL25 was significantly higher in adenocarcinomas (ACs) compared to squamous cell carcinomas (SCCs) (p = 0.04, and p< 0.0001). Similar to tissues, AC and SCC cell lines were positive for CCR9 expression. Despite of marginal difference in CCR9 expression, AC cells showed higher migratory and invasive potential in response to CCL25, compared to SCC cells. This differential biological response of AC cells was primarily due to differential expression of matrix metalloproteinases and tissue inhibitor of metalloproteinases under the influence of CCL25. Our results suggest CCR9 as a potential target for developing new treatment modality for NSCLC. Additionally, differential serum CCL25 level in ACs and SCCs, two NSCLC subtypes, suggest its potential as a non-invasive diagnostic/prognostic biomarker.

Downregulation of ribosomal protein S6 inhibits the growth of non-small cell lung cancer by inducing cell cycle arrest, rather than apoptosis

Ribosomal protein S6 (rpS6), a component of the small 40S ribosomal subunit, has been found to be associated with multiple physiological and pathophysiological functions. However, its effects and mechanisms in non-small cell lung cancer (NSCLC) still remain unknown. Here, we showed that expressions of total rpS6 and phosphorylation rpS6 (p-rpS6) were both significantly overexpressed in NSCLC. Further survival analysis revealed the shortened overall survival (OS) and relapse-free survival (RFS) in p-rpS6 overexpressed patients and confirmed it as an independent adverse predictor. Stable downregulation of rpS6 in lung adenocarcinoma A549 and squamous cell carcinoma H520 cell lines was then achieved by two specific small hairpin RNA (shRNA) lentiviruses separately. Subsequent experiments showed that downregulation of rpS6 dramatically inhibited cell proliferation in vitro and tumorigenicity in vivo. Moreover, loss of rpS6 promoted cells arrested in G0-G1 phase and reduced in G2-M phase, along with the expression alterations of relative proteins. However, no notable change in apoptosis was observed. Collectively, these results suggested that rpS6 is overactivated in NSCLC and its downregulation suppresses the growth of NSCLC mainly by inducing G0-G1 cell cycle arrest rather than apoptosis.