Synergistic effect of gefitinib and rofecoxib in mesothelioma cells

<p>Abstract</p> <p>Background</p> <p>Malignant mesothelioma (MM) is an aggressive tumor that is resistant to conventional modes of treatment with chemotherapy, surgery or radiation. Research into the molecular pathways involved in the development of MM should yield information that will guide therapeutic decisions. Epidermal growth factor receptor (EGFR) and cyclooxygenase-2 (COX-2) are involved in the carcinogenesis of MM. Combination of COX-2 and EGFR inhibitors, therefore, could be an effective strategy for reducing cell growth in those lines expressing the two molecular markers.</p> <p>Results</p> <p>In order to verify the effect of COX-2 and EGFR inhibitors, five MM cell lines NCI-2452, MPP89, Ist-Mes-1, Ist-Mes-2 and MSTO-211 were characterized for COX-2 and EGFR and then treated with respective inhibitors (rofecoxib and gefitinib) alone and in combination. Only MPP89, Ist-Mes-1 and Ist-Mes-2 were sensitive to rofecoxib and showed growth-inhibition upon gefitinib treatment. The combination of two drugs demonstrated synergistic effects on cell killing only in Ist-Mes-2, the cell line that was more sensitive to gefitinib and rofecoxib alone. Down-regulation of COX-2, EGFR, p-EGFR and up-regulation of p21 and p27 were found in Ist-Mes-2, after treatment with single agents and in combination. In contrast, association of two drugs resulted in antagonistic effect in Ist-Mes-1 and MPP89. In these cell lines after rofecoxib exposition, only an evident reduction of p-AKT was observed. No change in p-AKT in Ist-Mes-1 and MPP89 was observed after treatment with gefitinib alone and in combination with rofecoxib.</p> <p>Conclusions</p> <p>Gefitinib and rofecoxib exert cell type-specific effects that vary between different MM cells. Total EGFR expression and downstream signalling does not correlate with gefitinib sensitivity. These data suggest that the effect of gefitinib can be potentiated by rofecoxib in MM cell lines where AKT is not activated.</p

Cell-to-Cell Signaling Influences the Fate of Prostate Cancer Stem Cells and Their Potential to Generate More Aggressive Tumors

An increasing number of malignancies has been shown to be initiated and propelled by small subpopulations of cancer stem cells (CSC). However, whether tumor aggressiveness is driven by CSC and by what extent this property may be relevant within the tumor mass is still unsettled. To address this issue, we isolated a rare tumor cell population on the basis of its CD44+CD24− phenotype from the human androgen-independent prostate carcinoma cell line DU145 and established its CSC properties. The behavior of selected CSC was investigated with respect to the bulk DU145 cells. The injection of CSC in nude mice generated highly vascularized tumors infiltrating the adjacent tissues, showing high density of neuroendocrine cells and expressing low levels of E-cadherin and β-catenin as well as high levels of vimentin. On the contrary, when a comparable number of unsorted DU145 cells were injected the resulting tumors were less aggressive. To investigate the different features of tumors in vivo, the influence of differentiated tumor cells on CSC was examined in vitro by growing CSC in the absence or presence of conditioned medium from DU145 cells. CSC grown in permissive conditions differentiated into cell populations with features similar to those of cells held in aggressive tumors generated from CSC injection. Differently, conditioned medium induced CSC to differentiate into a cell phenotype comparable to cells of scarcely aggressive tumors originated from bulk DU145 cell injection. These findings show for the first time that CSC are able to generate differentiated cells expressing either highly or scarcely aggressive phenotype, thus influencing prostate cancer progression. The fate of CSC was determined by signals released from tumor environment. Moreover, using microarray analysis we selected some molecules which could be involved in this cell-to-cell signaling, hypothesizing their potential value for prognostic or therapeutic applications

Factors to Consider for the Correct Use of γH2AX in the Evaluation of DNA Double-Strand Breaks Damage Caused by Ionizing Radiation

People exposed to ionizing radiation (IR) both for diagnostic and therapeutic purposes is constantly increasing. Since the use of IR involves a risk of harmful effects, such as the DNA DSB induction, an accurate determination of this induced DNA damage and a correct evaluation of the risk–benefit ratio in the clinical field are of key relevance. γH2AX (the phosphorylated form of the histone variant H2AX) is a very early marker of DSBs that can be induced both in physiological conditions, such as in the absence of specific external agents, and by external factors such as smoking, heat, background environmental radiation, and drugs. All these internal and external conditions result in a basal level of γH2AX which must be considered for the correct assessment of the DSBs after IR exposure. In this review we analyze the most common conditions that induce H2AX phosphorylation, including specific exogenous stimuli, cellular states, basic environmental factors, and lifestyles. Moreover, we discuss the most widely used methods for γH2AX determination and describe the principal applications of γH2AX scoring, paying particular attention to clinical studies. This knowledge will help us optimize the use of available methods in order to discern the specific γH2AX following IR-induced DSBs from the basal level of γH2AX in the cells

Anterior Capsule of the Lens: Comparison of Morphological Properties and Apoptosis Induction following FLACS and Standard Phacoemulsification Surgery

Purpose. Comparative evaluation of morphological features of anterior capsules and apoptosis induction in epithelial cells after femtosecond laser-assisted cataract surgery (FLACS) and standard phacoemulsification surgery. Methods. Group 1: 30 FLACS anterior capsulotomies and Group 2: 30 manual anterior continuous curvilinear capsulorhexes. All patients were operated on by the same experienced surgeon. Morphological features of the anterior capsules and apoptosis induction in epithelial cells were evaluated. Results. All patients revealed a significant mean best-corrected visual acuity (BCVA) improvement 3 months after surgery, and no major intraoperative nor postoperative complications occurred. The capsular epithelium appeared to be preserved in both groups. Scanning electron microscopy analysis revealed irregular saw-tooth shaped edges in capsules from Group 1 whereas capsules from Group 2 showed regular and smooth edges. A statistically significant higher expression of the downstream apoptotic effector cleaved caspase 3 was observed in Group 1. Conclusions. The saw-tooth appearance was likely due to the progressive sequence of laser pulses on the capsule. The low energy/high frequency properties of the laser pulse, combined with an overlapped pulse pattern, resulted in highly continuous morphology of capsule edges. The higher apoptosis induction in FLACS group might be due to photodisruption-dependent plasma generation and formation of cavitation bubbles

Metabolic polymorphisms and urinary biomarkers in subjects with low benzene exposure

The effect of some common metabolic polymorphisms on the rate of trans,trans-muconic acid (TMA) and S-phenylmercapturic acid (SPMA) excretion was investigated in 169 policemen exposed to low benzene levels (g/m(3)) during the work shift. End-shift urinary concentrations of TMA and SPMA, normalized to unmetabolized blood benzene concentration, were used as indicators of individual metabolic capacity. CYP2E1, NQO1, GSTM1, and GSTT1 polymorphisms were analyzed in all subjects by polymerase chain reaction (PCR)-restriction fragment length (RFL). The results obtained show significantly elevated levels of TMA and SPMA in urine of smokers compared to nonsmokers, whereas no correlation with environmental benzene was observed. TMA/blood benzene ratio was partially modulated by glutathione S-transferase (GST) genotypes, with significantly higher values in null individuals (GSTM1 and GSTT1 combined). However, a greater fraction of total variance of TMA/blood benzene in the study population was explained by other independent variables, that is, season of sampling, smoking habits, and gender. Variance in SPMA/blood benzene ratio was only associated with smoking and occupation, whereas no significant role was observed for the metabolic polymorphisms considered. These results suggest that in a population exposed to very low benzene concentrations, urinary TMA and SPMA levels are affected to a limited extent by metabolic polymorphisms, whereas other factors, such as gender, lifestyle, or other confounders, may account for a larger fraction of the interindividual variability of these biomarkers

Effects of parthenolide on the expression of pro-inflammatory genes.

<p>LNCaP (<b>A</b>), PC3 (<b>B</b>), DU145 (<b>C</b>) cells were kept in normoxia and exposed to 1% O₂ in the presence and absence of 5 µM parthenolide. mRNA levels for VEGF, RAGE, P2X7R, PTX3, COX2, CXCR4 and HO1 were analyzed by real-time PCR, normalized to the housekeeping gene 18S rRNA and expressed as fold induction with respect to the normoxic untreated control (set at 1). For each gene we present the effect of parthenolide at the time points where hypoxia exerted a maximum increase on its transcription. *Hypoxic parthenolide treated cells <i>vs</i> hypoxic cells. C: control normoxic untreated cells. P: normoxic parthenolide treated cells. H: hypoxic cells. HP: parthenolide treated hypoxic cells.</p

Effects of Parthenolide on HIF1α and HIF3α hypoxia dependent activation.

<p>A LNCaP, DU145 and PC3 cells were kept in normoxia and exposed to 1% O₂ for 4 h in the presence and absence of 5 µM parthenolide. HIF1α content was measured in the nuclear fraction by immunoblot analysis. β-actin was used as a loading control. For each gene a representative experiment is shown. Mean densitometry of HIF1α relative to β-actin is also evidenced and expressed as arbitrary units. <b>B</b> DU145 cells were incubated under normoxic or hypoxic conditions for 48 h in the presence and absence of 5 µM parthenolide. HIF3α content was measured in the nuclear fraction by immunoblot analysis. β-actin was used as a loading control. For each gene a representative experiment is shown. Mean densitometry of HIF3α relative to β-actin is also evidenced and expressed as arbitrary units. Mean values ± SE. C: control normoxic untreated cells. P: normoxic parthenolide treated cells. H: hypoxic cells. HP: parthenolide treated hypoxic cells. *Hypoxic parthenolide treated cells <i>vs</i> hypoxic cells.</p

Effects of parthenolide on the expression of the anti-inflammatory gene HO1.

<p>LNCaP, PC3 and DU145 cells were kept in normoxia and exposed to 1% O₂ in the presence and absence of 5 µM parthenolide. mRNA levels for HO1 were analyzed by real-time PCR, normalized to the housekeeping gene 18S rRNA and expressed as fold induction with respect to the normoxic untreated control (set at 1). We show the effect of parthenolide after 4 h treatment when the drug maximally upregulated HO1 expression in normoxic cultures and after 24 h (LNCaP, DU145) and 48 h (PC3) stimulation when hypoxia exerted a maximum increase on its transcription. Mean values ± SE. * Normoxic and hypoxic parthenolide treated cells <i>vs</i> normoxic control cells. C: control normoxic untreated cells. P: normoxic parthenolide treated cells. H: hypoxic cells. HP: parthenolide treated hypoxic cells.</p

Hypoxic regulation of NF-kB nuclear expression in LNCaP, DU145 and PC3 cells.

<p>Cell were exposed to 1% O₂ from 0.5 up to 24 h or left untreated. After the times indicated, cells were processed and the nuclear content of p50 and p65 was detected by immunoblot analysis. β-actin was used as a loading control. A representative experiment for each gene in all the studied cell lines is shown. Mean densitometry of NF-kB p50 and p65 relative to β-actin ± SE is also evidenced and expressed as arbitrary units. * Hypoxic cells <i>vs</i> normoxic control cells.</p