Variety Of Carcinoma Cell Lines Research Articles

Combined proteomic and transcriptomic approaches reveal externalized keratin 8 as a potential therapeutic target involved in invasiveness of head and neck cancers.

Keratin 8 (K8) expressed at the surface of cancer cells, referred as externalized K8 (eK8), has been observed in a variety of carcinoma cell lines. K8 has been previously reported to be expressed in poorly differentiated head and neck squamous cell carcinoma (HNSCC); however, its role during the invasive phase of upper aerodigestive tract tumorigenesis is unknown. Cohorts of HNSCC tumors for protein and mRNA expression and panel of cell lines were used for investigation. K8 was found to be externalized in a majority of HNSCC cell lines. Among the two main K8 protein isoforms only the 54 kDa was found to be present at the plasma membrane of HNSCC cells. The plasminogen-induced invasion of HNSCC cells was inhibited by the anti-eK8 D-A10 antagonist monoclonal antibody. Overexpression of K8 mRNA and protein were both correlated with tumor aggressive features and poor outcome. The effect of eK8 neutralization on invasion, its presence exclusively in cancer cells and the association of K8 expression with aggressive features and poor clinical outcome in HNSCC unravel eK8 as key player in invasion and a promising therapeutic target in HNSCC.

Spotlight on Volasertib: Preclinical and Clinical Evaluation of a Promising Plk1 Inhibitor.

Considering the important side effects of conventional microtubule targeting agents, more and more research focuses on regulatory proteins for the development of mitosis-specific agents. Polo-like kinase 1 (Plk1), a master regulator of several cell cycle events, has arisen as an intriguing target in this research field. The observed overexpression of Plk1 in a broad range of human malignancies has given rise to the development of several potent and specific small molecule inhibitors targeting the kinase. In this review, we focus on volasertib (BI6727), the lead agent in category of Plk1 inhibitors at the moment. Numerous preclinical experiments have demonstrated that BI6727 is highly active across a variety of carcinoma cell lines, and the inhibitor has been reported to induce tumor regression in several xenograft models. Moreover, volasertib has shown clinical efficacy in multiple tumor types. As a result, Food and Drug Administration (FDA) has recently awarded volasertib the Breakthrough Therapy status after significant benefit was observed in acute myeloid leukemia (AML) patients treated with the Plk1 inhibitor. Here, we discuss both preclinical and clinical data available for volasertib administered as monotherapy or in combination with other anticancer therapies in a broad range of tumor types.

Synthesis and biological evaluation of novel pyridine derivatives as potential anticancer agents and phosphodiesterase-3 inhibitors

Phosphodiesterases (PDEs) have been studied in a variety of tumours; data have suggested that the levels of PDE activities are elevated and, therefore, the ratios of cGMP to cAMP are affected. In addition, PDE inhibitors are potential targets for tumour cell growth inhibition and induction of apoptosis. Nonselective PDE inhibitors, such as theophylline or aminophylline, are known regulators of growth in a variety of carcinoma cell lines, suggesting a potential role for PDE inhibitors as anticancer drugs. In the current study, we reported the synthesis of novel derivatives of 6-aryl-4-imidazolyl-2-imino-1,2-dihydropyridine-3-carbonitriles (Ia,b,c) and their 2-oxo isosteres (IIa,b,c,d). All the compounds were evaluated for their PDE3A inhibitory effects, as well as their cytotoxic effects on MCF-7 and HeLa cell lines. Moreover, structure-activity relationships were studied. 4-(1-benzyl-2-ethylthio-5-imidazolyl)-6-(4-bromophenyl)-2-imino-1,2-dihydropyridine-3-carbonitrile (Ib) exhibited the strongest PDE3A inhibitory effects with an IC50 of 3.76±1.03nM. Compound Ib also showed the strongest cytotoxic effects on both the HeLa and MCF-7 cells with an IC50 of 34.3±2.6μM and 50.18±1.11μM, respectively. There was a direct correlation between PDE3 inhibition and anticancer activity for the synthesised compounds. The data reported here support our view that PDEs represent promising cellular targets for antitumor treatment.

Abstract 5346: A novel immunotoxin comprising quadruple RNase tethered to an internalizing anti-TROP-2 humanized MAb shows potent cytotoxicity against diverse solid tumors in vitro

Abstract Ribonucleases (RNase) are potential anti-tumor agents, and their potency can be enhanced by recombinant fusion or chemical conjugation with tumor-targeting antibodies, as have been demonstrated for ranpirnase (Rap), an amphibian RNase, conjugated to LL2, a murine anti-CD22 MAb (Newton et al., Blood 2001; 97: 528-35) or fused to a humanized anti-CD74 MAb (Chang et al., Blood 2005; 106: 4308-14). We have now applied the Dock-and-Lock (DNL) method to link four copies of Rap site-specifically to the carboxyl termini of a panel of tumor-targeting humanized antibodies, in particular, hRS7, an internalizing, humanized, anti-TROP-2 MAb. We combined Rap-DDD2, a fusion protein comprising Rap and the dimerization and docking domain (DDD) of cAMP-dependent protein kinase (PKA), with hRS7-IgG-AD2, a fusion protein comprising two AD2 peptides derived from A-kinase anchoring proteins (AKAPs) having each AD2 appended to the carboxyl terminus of the heavy chain. Since Rap-DDD2 is isolated as a dimer, the resulting DNL complex, designated E1-Rap, contains four copies of Rap tethered to a bivalent hRS7 IgG. In the present study, we compared the in vitro growth inhibitory properties of E1-Rap against a variety of carcinoma cell lines. In breast (MDA-MB-468), cervical (ME-180), and pancreatic (BxPC-3 and Capan-1) tumor lines, all of which express high levels of TROP-2, E1-Rap was very potent, showing EC50 in the subnanomolar range (5 to 890 pM), which was 1,000- to 100,00-fold higher than untargeted Rap or the combination of Rap and hRS7. In cell lines expressing moderate levels of TROP-2, such as the three prostate cancer lines (PC-3, DU 145, and LNCaP), E1-Rap was less potent, but still showed EC50 in the nanomolar range (1 to 890 nM). The cell binding data obtained for these solid cancer cell lines suggest that the sensitivity of a cell line to E1-Rap appears to correlate with its TROP-2 expression on the cell surface. No toxicity was observed for E1-Rap in the prostate cancer line, 22Rv1, which fails to bind hRS7. Based on these results, further in vivo studies are warranted to better determine the efficacy of E1-Rap for future development as a new candidate therapeutic for TROP-2-positive solid tumors, including breast, cervical, pancreatic, and prostatic carcinomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5346.

Activation of Nonsteroidal Anti-Inflammatory Drug-Activated Gene-1 via Extracellular Signal-Regulated Kinase 1/2 Mitogen-Activated Protein Kinase Revealed a Isochaihulactone-Triggered Apoptotic Pathway in Human Lung Cancer A549 Cells

The novel lignan isochaihulactone inhibits cell proliferation and is an effective inducer of apoptosis in a variety of carcinoma cell lines. To determine the mechanisms underlying these effects, we examined isochaihulactone-induced changes in gene expression using oligodeoxynucleotide-based microarray screening of a human lung carcinoma cell line, A549. Isochaihulactone-inducible genes included the early growth response gene-1 (EGR-1) and nonsteroidal anti-inflammatory drug-activated gene (NAG-1). Isochaihulactone increased EGR-1 and then NAG-1 mRNA and protein expression. Pure isochaihulactone induced phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Isochaihulactone-induced increases in EGR-1 and NAG-1 expression were reduced by the mitogen-activated protein kinase kinase 1/2 inhibitor 2'-amino-3'-methoxyflavone (PD98059), and this effect was not blocked by the phosphatidylinositol 3-kinase/protein kinase B pathway inhibitor 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002). Inhibition of isochaihulactone-induced NAG-1 expression by EGR-1 small interfering RNA blocked isochaihulactone-induced apoptosis in A549 cells, suggesting that induction of EGR-1 expression decreases survival of A549 cells. RNA interference using double-stranded RNA specific for NAG-1 also inhibited isochaihulactone-induced apoptosis, and cells transfected to increased NAG-1 expression had a greater apoptotic response to isochaihulactone and reduced colony formation efficiency. In addition, treatment of nude mice with isochaihulactone increased the in vivo NAG-1 expression as examined by immunohistochemistry from tumor biopsy. Isochaihulactone treatment increased the luciferase activity of NAG-1 in A549 cells transfected with the NAG-1 promoter construct. This induction increased expression of NAG-1 that was p53-independent and Sp1-dependent. Our findings suggest that NAG-1 expression is up-regulated by isochaihulactone through an ERK-dependent pathway involving the activation of EGR-1.

Up-regulation of the enzymes involved in prostacyclin synthesis via Ras induces vascular endothelial growth factor

The constitutive activation of Ras is an important step in the development and progression of several different cancers and is known to increase the level of cyclooxygenase 2 (COX-2). Prostaglandins are the downstream bioactive lipid mediators produced by the COX-2 enzyme. We sought to determine the role of Ras-induced up-regulation of the enzymes involved in prostacyclin biosynthesis in nontransformed rat intestinal epithelial cells (IECs). Messenger RNA (mRNA) and protein expression were analyzed by Northern and Western analysis, respectively, to determine the level of enzymes induced by Ras. In vitro assays were used to determine the production of vascular endothelial growth factor (VEGF) and prostaglandins as well as the promoter and enzymatic activation of the rate-limiting enzyme in prostaglandin production (phospholipase A(2) [cPLA(2)]). The inducible expression of Ha-Ras(V12) increased the production of prostaglandin (PG)F(2alpha) and prostacyclin by 2- and 13-fold, respectively. The induction of Ha-Ras(V12) also up-regulated the mRNA and protein levels of cPLA(2), COX-2, and prostacyclin synthase, as well as the promoter and enzyme activity of cPLA(2). Furthermore, oncogenic Ras increased the production of the pro-angiogenic factor VEGF. The increase of VEGF was abolished after treatment with celecoxib, a selective COX-2 inhibitor. The addition of PGI 2 alone also induced the expression of VEGF. Inducible Ha-Ras(V12) increases the production of PGI(2) through the coordinate up-regulation of cPLA(2), COX-2, and prostacyclin synthase (PGIS). The production of PGI(2) leads to an increase in the level of the pro-angiogenic factor VEGF, which is known to play a crucial role in the regulation of tumor-associated angiogenesis.

Phosphodiesterase inhibitors as anti-cancer drugs

It is well known that high intracellular levels of cAMP can effectively kill cancer cells in vitro. Unfortunately substances elevating cAMP such as forskolin, 8-bromo-cAMP, 8-chloro-cAMP, monobutiryl or dibutiryl cAMP are not recommended to be used as anti-cancer drugs because of their high cytotoxicity. In contrast blockers of phosphodieterases such as theophylline and aminophylline, which could elevate intracellular cAMP, are commonly used as anti-asthma drugs reaching concentrations in the blood of 10-20 microg/ml. We tested the effectiveness of theophylline and aminophylline to induce cell death alone or in combination with common anti-cancer drugs such as cisplatin and gemcitabine (gemzar). We examined such drug combinations in the induction of cell death in a variety of carcinoma cell lines derived from human ovarian, prostate and lung cancer and in granulosa cell line transformed by SV40 and Ras oncogene. While theophylline could induce moderate cell death alone, at 20-25 microg/ml concentrations, aminophylline was ineffective at this concentration. Theophylline (at 15-25 ng/ml) was found in all four representative cell lines to synergize with gemcitabine or cisplatin to induce programmed cell death, which permits a reduction in the effective doses of cisplatin and gemcitabine by 2-3-fold. The effect of theophylline in induction of apoptosis involved reduction of intracellular levels of Bcl2. Such a reduction was proportional to the extent of apoptosis induced by theophylline as well as by the combined drug treatments. Therefore, we propose that theophylline should be considered as a potential anti-cancer drug in combination with other chemotherapeutic drugs. Screening of other phosphodiesterase blockers, which are not severely toxic, could open a possibility to improved chemotherapeutic cancer treatments with reduced undesired side-effects. A clinical trial, using theophylline as an anti-cancer drug, is currently being conducted in lung cancer patients.

Tumour-expressed CD43 (sialophorin) mediates tumourmesothelial cell adhesion.

Mesothelial cell intercellular adhesion molecule-1 (ICAM-1) has recently been shown to play a role in tumour cell adherence to the peritoneum. However, solid tumours poorly express its most ubiquitous ligand, beta2 integrin. The aim of this study was to investigate the role of the beta2 integrin subunit and CD43, a known ligand for ICAM-1, in the development of peritoneal metastases. beta2 Integrin subunit and CD43 expression was assessed on a number of tumour cell lines. Adhesion of SW1222 and PSN-1 cells to human peritoneal mesothelial cells was investigated using a fluorometric assay incorporating an inhibitory antibody to beta2 integrin and CD43. beta2 Integrin expression was not inducible on these tumour cell lines, but Western blotting demonstrated CD43 expression in all the cancer cell lines examined and cell surface expression was confirmed by flow cytometry. The anti-CD43 antibody significantly reduced adhesion of PSN-1 and SW1222 cells to HPMC, however beta2 integrin inhibition did not reduce tumour cell adhesion. CD43 is expressed by a variety of carcinoma cell lines, and plays a role in tumour cell-peritoneal adhesion probably via interactions with its putative ligand ICAM-1.

Modulation of Mucin Synthesis by γ-Interferon in Human Colon Adenocarcinoma Cells

Objectives: Recombinant human interferon has been shown to enhance the expression of histocompatibility antigens and certain tumor-associated antigens in a variety of carcinoma cell lines. Since many tumor-associated antigens are mucins, we investigated the effect of recombinant human γ-interferon on mucin production and secretion by colon cancer cell lines. Methods: Control and γ-interferon-treated cells were labeled with [³H]glucosamine in DMEM-H16 medium with 5% FCS for 24 h. Analysis was performed by gel filtration on Sepharose CL-4B columns, and the high-molecular-weight glycoprotein eluted at the void volume from the cytosol and medium was counted (expressed as dpm/4 × 10⁶ cells). In another experiment the void volume was compared to concentration curves of standard mucins (expressed as mg/10⁷ cells). Results: γ-Interferon increased mucin synthesis in HT-29 and LIM-6 cells, but not in LS174T and CACO2 cells. In HT-29 and LIM-6 cells, mucin synthesis was induced by γ-interferon in a dose-dependent manner. The percentage of mucin secreted into the medium was also increased. Conclusion: The heterogeneity of response of human colon cancer cell lines to γ-interferon by mucin production may limit the specific role of γ-interferon as a modulator of mucin-type tumor-associated antigens.

Induction of Egr-1 expression by the retinoid AHPN in human lung carcinoma cells is dependent on activated ERK1/2.

The novel retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN/CD437) inhibits cell proliferation and is a very effective inducer of apoptosis in a variety of carcinoma cell lines. In order to obtain greater insight into the mechanism of AHPN-induced growth arrest and apoptosis, we began to examine AHPN-induced changes in gene expression by cDNA array screening using human lung carcinoma H460 cells. This analysis identified several AHPN-inducible genes, including the immediate-early genes Egr-1 and Nur77. AHPN was able to increase Egr-1 and Nur77 mRNA expression and protein in a variety of carcinoma cell lines. This induction appeared to be regulated at the transcriptional level and was specific for AHPN since an RAR- and an RXR-selective retinoid were inactive. These results suggest that the induction of Egr-1 and Nur77 by AHPN is independent of nuclear retinoid receptors and involves a novel mechanism. Overexpression of Bcl-2, which inhibits AHPN-induced apoptosis but not growth arrest in human T cell lymphoma Molt-4 cells, did not block the induction of immediate-early gene expression. Treatment of H460 cells with AHPN induced activation of the p38 MAP-kinase but not the ERK1/2 signaling pathway. However, inhibition of the ERK1/2 signaling pathway by PD98059 blocked the induction of Egr-1 and Nur77 mRNA while the p38 MAPK inhibitor PD169316 had little effect. Expression of a dominant-negative ERK1 completely abolished the increase in Egr-1 mRNA. Treatment with MAPK inhibitors or expression of dnERK1 reduced but did not block AHPN-induced apoptosis. Our results suggest that the induction of Egr-1 in H460 by AHPN requires active ERK1/2 and is independent of p38 activation. Egr-1, in cooperation with several other growth-suppressor proteins, is likely involved in AHPN-induced inhibition of cell growth and cell death.

HER4 Expression Correlates with Cytotoxicity Directed by a Heregulin-Toxin Fusion Protein

We have constructed, expressed, and purified a fusion protein, HAR-TX beta 2, consisting of heregulin-beta 2 fused to a binding-defective form of Pseudomonas exotoxin A, PE40. The fusion protein was found to induce receptor tyrosine phosphorylation in CEM cells transfected with HER4 alone or in combination with HER2 but not in cells transfected with HER2 or HER1 alone. The phosphorylation of receptor tyrosines was both dose-dependent and saturable in amounts similar to those shown to be active for native heregulin. HAR-TX beta 2 was specifically cytotoxic toward a variety of carcinoma cell lines in the ng/ml range. However, some tumor cell lines were found to be insensitive to the cytotoxic action of the fusion protein even at > 2 micrograms/ml. Relative amounts of HER4, HER3, and HER2 were determined on seven cell lines sensitive and four cell lines insensitive to HAR-TX beta 2. All lines that express HER4 were killed by HAR-TX beta 2, while none lacking HER4 were affected. HAR-TX beta 2 was able to bind to and signal via tyrosine phosphorylation in cell lines that co-express HER2 and HER3 in the absence of HER4 without inducing cytotoxicity. Thus HAR-TX beta 2 may prove to be a useful reagent for the targeting and elimination of HER4-positive tumor cells.

Characterization of a monoclonal antibody-defined human melanoma-associated antigen susceptible to induction by immune interferon.

To develop monoclonal antibodies (mAb) recognizing human melanoma-associated antigens (MAA) susceptible to modulation by immune interferon (IFN-gamma), hybridomas were constructed with splenocytes from a BALB/c mouse immunized with IFN-gamma-treated melanoma cells Colo 38. Screening of supernatants with control and IFN-gamma-treated melanoma cells showed that the mAb CL203 and CL207 display preferential reactivity with IFN-gamma-treated melanoma cells. The two mAb recognize the same (or spatially close) determinant on a 96,000 MAA which has a density of 0.36 X 10(6) antigenic sites/cell on untreated melanoma cells Colo 38 and of 1.39 X 10(6) and 1.54 X 10(6) on melanoma cells Colo 38 treated with IFN-gamma (final concentration, 200 U/ml) for 24 and 48 hr, respectively. The effect of IFN-gamma on the 96,000 MAA is dose- and time-dependent, reversible, and blocked by inhibitors of RNA and protein synthesis. Furthermore, the effect of IFN-gamma on the induction of the 96,000 MAA appears to be specific, inasmuch as IFN-alpha and IFN-beta do not induce the expression of the 96,000 MAA. The latter is also induced by IFN-gamma in a variety of carcinoma cell lines, but its level is markedly lower than on melanoma cells. Furthermore, the apparent m.w. of the antigen synthesized by the carcinoma cell lines in the presence of IFN-gamma ranges between 93,000 and 96,000. This molecular heterogeneity appears to reflect differences in the degree of glycosylation of the polypeptide moiety because the antigen synthesized by a variety of cell lines in the presence of tunicamycin has an apparent m.w. of 51,000.