Immunohistochemical assessment of protein phosphorylation state: the dream and the reality

The development of phosphorylation state-specific antibodies (PSSAs) in the 1980s, and their subsequent proliferation promised to enable in situ analysis of the activation states of complex intracellular signaling networks. The extent to which this promise has been fulfilled is the topic of this review. I review some applications of PSSAs primarily in the assessment of solid tumor signaling pathway activation status. PSSAs have received considerable attention for their potential to reveal cell type-specific activation status, provide added prognostic information, aid in the prediction of response to therapy, and most recently, demonstrate the efficacy of kinase-targeted chemotherapies. However, despite some successes, many studies have failed to demonstrate added value of PSSAs over general antibody immunohistochemistry. Moreover, there is still a large degree of uncertainty about the interpretation of complex and heterogeneous staining patterns in tissue samples and their relationship to the actual phosphorylation states in vivo. The next phase of translational research in applications of PSSAs will entail the hard work of antibody validation, gathering of detailed information about epitope-specific lability, and implementation of methods for standardization

Epigenetics of lung cancer

Epigenetics is the study of heritable changes in gene expression that occur without changes in DNA sequence. It has a role in determining when and where a gene is expressed during development. Perhaps the most well known epigenetic mechanism is DNA methylation whereby cytosines at position 5 in CpG dinucleotides are methylated. Histone modification is another form of epigenetic control, which is quite complex and diverse. Histones and DNA make up the nucleosome which is the structural unit of chromatin which are involved in packaging DNA. Apart from the crucial role epigenetics plays in embryonic development, transcription, chromatin structure, X chromosome inactivation and genomic imprinting, its role in an increasing number of human diseases is more and more recognized. These diseases include cancer, and lung cancer in particular has been increasingly studied for the potential biological role of epigenetic changes with the promise of better and novel diagnostic and therapeutic tools

Epidermal growth factor receptor inhibitors: A new prospective in the treatment of lung cancer

Lung cancer is the leading cause of death worldwide. Current treatment modalities, including chemotherapy, radiotherapy and surgery, provide only limited improvement in the natural course of this disease. Therefore, the development of new therapeutic strategies is highly awaited. This review focuses on recent achievements on a novel class of anticancer drugs targeting the EGFR (Epidermal Growth Factor Receptor). The EGFR family is a group of four structurally similar growth factor receptors with tyrosine-kinase activity (EGFR, HER2/neu, ErbB-3, ErbB-4), which dimerize upon binding with a number of ligands, including EGF (Epidermal Growth Factor) and TGF (Transforming Growth Factor), allowing downstream transduction of mitogenic signals. Overexpression of EGFR and HER2 is frequently found in non-small-cell lung cancer (NSCLC), which accounts for over 80% of all malignant lung tumors, and has been associated with a worse clinical outcome. New agents developed to inhibit EGFR function include monoclonal antibodies and small-molecule receptor tyrosine-kinase inhibitors. In this review, results of most recent clinical with EGFR inhibitors including monoclonal antibodies, such as Trastuzumab (Herceptin), IMC-C225 (Cetuximab) and others (ABX-EGF, EMD 72000), and tyrosine-kinase inhibitors, such as ZD1839 (Gefitinib, Iressa), OSI-774 (Erlotinib, Tarceva) and others (CI-1033, GW2016), are summarized. In particular, final results of phase II (IDEAL 1 and 2) and III (INTACT 1 and 2) studies of ZD1839 are reported. In IDEAL trials (ZD1839 single agent in patients pre-treated with chemotherapy) there was clear evidence of tumor regression, symptoms improvement and overall clinical benefit, whereas in the two INTACT trials (ZD1839 in combination with standard platinum-based chemotherapy in chemo-naive patients) ZD1839 did not improve either survival or other clinical endpoints. Possible explanations for these contradictory results and future perspectives are discussed. © 2004 Bentham Science Publishers Ltd

The role of systemic chemotherapy in the treatment of brain metastases from small-cell lung cancer

Brain is the most common site of metastatic spread in small-cell lung cancer (SCLC). Approximately 10% of SCLC patients have brain metastases (BM) already at diagnosis and an additional 40% will develop central nervous system (CNS) involvement during their disease course. Although whole brain radiotherapy and corticosteroids is considered the treatment of choice, accumulating evidence suggests that systemic chemotherapy may also play an important role. The concept of the brain as a pharmacologic sanctuary site for established metastases is in contrast with recent clinical observations of frequent BM responses with systemic chemotherapy. During the last decade, several reports about the effect of systemic chemotherapy on BM from SCLC have been published. Pooled data from five studies report 66% response rate (RR) in 64 patients with initial BM. In addition, an average RR of 36% is derived from five studies including 135 patients with delayed BM treated with systemic single agent chemotherapy. Among new drugs with activity in patients with SCLC brain metastases, camptothecin analog topotecan is one of the most promising with a 52% RR. Although whole brain radiation remains the standard treatment of established BM in SCLC there is an emerging role for systemic chemotherapy, particularly with the use of new active drugs as part of combined modality treatments

In vitro study of CI-994, a histone deacetylase inhibitor, in non-small cell lung cancer cell lines

CI-994 (N-acetyldinaline) is a novel oral compound with a wide spectrum of antitumor activity in preclinical models, in vitro and in vivo. The mechanism of action may involve inhibition of histone deacetylation and cell cycle arrest. We studied the action of CI-994 on two non-small cell lung cancer (NSCLC) cell lines: A-549 (adenocarcinoma) and LX-1 (squamous cell carcinoma). Different drug concentrations were tested, ranging from 0.01 to 160 μM at 24, 48, and 72 h of treatment, with MTT assay. A concentration-dependent cell survival inhibition was observed, with an IC50at 80 μM. The effect of CI-994, as demonstrated by recovery experiments, was cytostatic and seemed to be superimposable in both cell lines. Cytofluorimetric analysis to assess cell cycle perturbation and apoptosis was performed after 24 h of treatment, indicating a cell block with concomitant increase at G0G1phase, a reduction at S phase level at 20, 40, 80, and 160 μM, and apoptosis at the higher concentration (160 μM). When CI-994 was combined with antineoplastic agents commonly used in NSCLC management, a marked synergism of action (R = 1.8, R = 1.5) was observed between CI-994 (40 μM) and gemcitabine (0.01 μM) at 48 and 72 h of treatment. The same result was obtained with docetaxel (0.001 μM) combination (R = 1.4, R = 1.2), but no synergism of action was noted with paclitaxel. CI-994 showed no radiopotentiating effects, when combined with 100, 200, or 400 cGy irradiation. In conclusion, our experiments indicate that CI-994 is a promising novel cytostatic for the treatment of NSCLC. Its use in combination with standard anticancer agents, such as gemcitabine and docetaxel, is warranted. Copyright © 2005 Cognizant Comm. Corp

Pre-clinical evaluation of new antineoplastic agents in NSCLC cell lines: evidence of histological subtype-dependent cytotoxicity

The antiproliferative effect of paclitaxel, docetaxel, gemcitabine, topotecan, SN-38 and cis-platin was studied on 5 non-small cell lung cancer (NSCLC) cell lines, 3 of which were adenocarcinoma (ADK) and 2 squamous cell carcinoma (SCC). Cellular chemosensitivity was determined using the MTT in vitro assay after 48, 72 and 96 h of exposure to drug in concentration ranging from 0.001 to 100 microM. A concentration-dependent cell growth inhibition was observed for paclitaxel, gemcitabine, topotecan, SN-38 and cis-platin in all cell lines tested. Docetaxel showed a concentration-independent cytotoxicity and was 104 times more potent than cis-platin (IC50 = 0. 001 vs. 10 microM). Paclitaxel, gemcitabine, topotecan and SN-38 were 102 times more potent than cis-platin, with median IC50 = 0.1 microM at 72 h. The level of drug-induced cell growth inhibition appeared to be correlated, for some of the six drugs tested, with the tumor histological subtype. In particular, topotecan and cis-platin were more active in squamous cell carcinoma than in adenocarcinoma cell lines (p=0.006 and 0.001 respectively at 0.1 microM concentration), while paclitaxel was more active in ADK than in SCC cell lines (p=0.004 at 0.01 microM concentration). Ca-Lu-6, a cell line that, contrary to most other lung cancer cell lines, is wild-type for most oncogenes/tumor suppressor genes, was by far the most sensitive cell line used (p=0.002, 0.003, 0.01 for paclitaxel, topotecan and cis-platin respectively, at 1 microM concentration), showing a >50% growth inhibition to new drugs at a concentration of 0.01 microM. In conclusion, all these new compounds tested were found to be more potent than cis-platin in affecting cellular proliferation of six NSCLC cell lines studied. We suggest that the specific histological subtype and molecular pattern of the cell line being treated could affect the antiproliferative effect of these drugs

In vitro study of farnesyltransferase inhibitor SCH 66336, in combination with chemotherapy and radiation, in non-small cell lung cancer cell lines

K-ras alterations have been reported in 20-30% of non-small cell lung cancer (NSCLC) and represent a suitable target for the development of novel anticancer agents, such as Farnesyl transferase inhibitors (FTi), a new class of agents inhibiting the post-translational modification of the K-ras proteins. The effectiveness of FTi SCH66336 in inhibiting cell proliferation and deranging cell cycle of NSCLC cell lines as well as its interaction with chemotherapy or radiation have been evaluated. The activity of Fri SCH66336, alone or in combination with paclitaxel, gemcitabine, and radiotherapy, was examined in 3 cell lines, A-549, LX-1 and CaLu-6, by colorimetric MTT assay. Cell cycle perturbation and apoptosis were also assessed by cytofluorimetric analysis. The activity of SCH 66336 was found to be concentration- and time-dependent. The effect of SCH 66336, as demonstrated by cell,growth recovery experiments, resulted cytostatic and it was superimposable in both cell lines bearing 2 different K-ras mutations (A-549 and LX-1) and in K-ras wild-type Ca-Lu-6. In all cell lines the combination of SCH 66336 and paclitaxel resulted in a synergism of action when SCH 66336 followed paclitaxel treatment, whereas, antagonism was found when SCH 66336 preceded paclitaxel treatment. No significant synergism or addition with SCH 66336 followed by radiation treatment was noted. Different cell cycle phase blocks at various drug concentrations were observed. In conclusion, SCH 66336 displays concentration-dependent cytostatic antitumour activity and schedule-dependent synergy with 2 commonly used anticancer agents in NSCLC cell lines. Further clinical testing of these combinations is warranted

In vitro study of CI-994, a histone deacetylase inhibitor, in non-small cell lung cancer cell lines

none8CI-994 (N-acetyldinaline) is a novel oral compound with a wide spectrum of antitumor activity in preclinical models, in vitro and in vivo. The mechanism of action may involve inhibition of histone deacetylation and cell cycle arrest. We studied the action of CI-994 on two non-small cell lung cancer (NSCLC) cell lines: A549 (adenocarcinoma) and LX-1 (squamous cell carcinoma). Different drug concentrations were tested, ranging from 0.01 to 160 mu M at 24, 48, and 72 h of treatment, with MTT assay. A concentration-dependent cell survival inhibition was observed, with an IC50 at 80 mu M. The effect of CI-994, as demonstrated by recovery experiments, was cytostatic and seemed to be superimposable in both cell lines. Cytofluorimetric analysis to assess cell cycle perturbation and apoptosis was performed after 24 h of treatment, indicating a cell block with concomitant increase at G(0)/G(1) phase, a reduction at S phase level at 20, 40, 80, and 160 mu M, and apoptosis at the higher concentration (160 mu M). When CI-994 was combined with antineoplastic agents commonly used in NSCLC management, a marked synergism of action (R = 1.8, R = 1.5) was observed between CI-994 (40 mu M) and gemcitabine (0.01 mu M) at 48 and 72 h of treatment. The same result was obtained with docetaxel (0.001 mu M) combination (R = 1.4, R = 1.2), but no synergism of action was noted with paclitaxel. CI-994 showed no radiopotentiating effects, when combined with 100, 200, or 400 cGy irradiation. In conclusion, our experiments indicate that CI-994 is a promising novel cytostatic for the treatment of NSCLC. Its use in combination with standard anticancer agents, such as gemcitabine and docetaxel, is warranted.noneLoprevite M; Tiseo M; Grossi F; Scolaro T; Semino C; Pandolfi A; Favoni R; Ardizzoni ALoprevite M; Tiseo M; Grossi F; Scolaro T; Semino C; Pandolfi A; Favoni R; Ardizzoni