Functional and pharmacodynamic evaluation of metronomic cyclophosphamide and docetaxel regimen in castration-resistant prostate cancer.

AIM: The aim of our study was to investigate the association of docetaxel and metronomic cyclophosphamide (CYC) in castration-resistant prostate cancer (CRPC). MATERIALS & METHODS: CRPC xenografts were established with PC3 cells. Mice were treated with a combination of CYC (50 mg/kg/day) and docetaxel (10-30 mg/kg/week) or with docetaxel alone. Docetaxel plasma levels were analyzed in patients receiving the drug alone or combined with CYC. RESULTS: Metronomic CYC is an effective adjuvant in blocking tumor growth in vivo, with comparable efficacy and less toxic effects compared with docetaxel treatment. CYC acts by downregulating cell proliferation and inducing apoptosis thorough upregulation of p21 and inhibition of angiogenesis. Finally, CYC increases docetaxel plasma levels in patients. CONCLUSION: Metronomic CYC exerts anti-tumoral effects in an in vivo model of prostate cancer and in patients with CRPC, and also increases the bioavailability of docetaxel. These results explain the favorable toxicity and activity profiles observed in patients treated with this regimen.

Synergistic effect of gefitinib and rofecoxib in mesothelioma cells.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Molecular analysis of the effects of Piroxicam and Cisplatin on mesothelioma cells growth and viability.

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been proposed for prevention and treatment of a variety of human cancers. Piroxicam, in particular, has been recently shown to exert significant anti-tumoral activity in combination with cisplatin (CDDP) on mesothelioma cells. However, the mechanisms through which NSAIDs regulate the cell cycle as well as the signal pathways involved in the growth inhibition, remain unclear. In the present study, using two mesothelioma cell lines, MSTO-211H and NCI-H2452, we have investigated the influence of piroxicam alone and in association with CDDP on proliferation, cell cycle regulation and apoptosis. In both cell lines a significant effect on cell growth inhibition, respect to the control, was observed with all the drugs tested. Moreover, treatment with piroxicam or CDDP alone altered the cell cycle phase distribution as well as the expression of some cell cycle regulatory proteins in both cell lines. These effects were increased, even if in a not completely overlapping manner, after treatment with the association of piroxicam and CDDP. In particular, the two drugs in NCI cell line had a synergistic effect on apoptosis, probably through activation of caspase 8 and caspase 9, while the most evident targets among the cell cycle regulators were cyclin D1 and p21waf1. These results suggest that the association of piroxicam and CDDP specifically triggers cell cycle regulation and apoptosis in different mesothelioma cell lines and may hold promise in the treatment of mesothelioma.

Direct and indirect effects of Growth Hormone Deficiency (GHD) on lung function in children: A mediation analysis.

BACKGROUND: Studies on pulmonary function tests (PFTs) in Growth Hormone Deficiency (GHD) children are lacking. The aims of this study were: (i) to investigate PFTs in GHD pre-pubertal children with respect to Controls, before starting Growth Hormone Therapy (GHT) (T0); (ii) to evaluate changes of PFTs in GHD vs Controls, after 1-year GHT (T1). For both aims the mediation analysis (MA) was applied to evaluate the extent to which the relationship between GHD and PFTs could be ascribed to a height-mediated (indirect) or a GH direct effect. METHODS: 47 pre-pubertal GHD children (aged 5-14 years) underwent PFTs at T0 and T1. At T0, 47 healthy children matched for age and sex were enrolled as Controls. A MA was performed to assess the relationship between GHD and PFTs and height. Statistical analyses were performed using the statistical software R (https://cran.r-project.org/mirrors.html). A p-value <0.05 was considered significant. MEASUREMENTS AND MAIN RESULTS: At T0, PFTs indices were significantly lower in GHD than in Controls. From T0 to T1 a significant improvement was found in PFTs. The percentages of the mediated effect on FVC, FEV1, FEF25-75% and TLC were <50% at T0, suggesting that the direct effect was prevalent. At T1, the percentages of the mediated effect for spirometry indices were ≥50%, indicating that the indirect (height-mediated) effect was the most relevant. CONCLUSIONS: The study shows that pre-pubertal children with GHD have an impairment of lung function not exclusively attributable to the indirect (height-mediated) effect, but also to the direct GH action which is mitigated after 1-year of GHT.

Evaluation of antitumoral properties of the protease inhibitor indinavir in a murine model of hepatocarcinoma.

PURPOSE: Accumulating evidences show a higher incidence of hepatic neoplasm in HIV/hepatitis C virus (HCV)-coinfected individuals compared with HCV-monoinfected patients. Treatment with HIV-1 protease inhibitors inhibited cancer-promoted angiogenesis in HIV-infected patients affected by Kaposi sarcoma. We aimed to evaluate the antineoplastic potential activities of the protease inhibitor indinavir (Crixivan) in in vitro and in vivo hepatocarcinoma models. EXPERIMENTAL DESIGN: We analyzed effects of indinavir on cell growth and invasiveness in Huh7 and SK-HEP-1 hepatocarcinoma cell lines and on in vivo tumor growth of the same cells in nude mice. Morphologic and molecular analyses on explanted tumors were carried out to evaluate vascularization and apoptosis. RESULTS: We observed a reduced ability to invade an in vitro extracellular matrix for both cell lines treated with indinavir compared with controls (P = 0,001). Moreover, indinavir treatment was able to inhibit matrix metalloproteinase-2 proteolytic activation, whereas there was no effect on cell proliferation. The drug was also able to delay in vivo tumor growth. The inhibition of tumor growth was statistically significant from days 6 to 21 (P = 0.004 and P = 0.003, respectively). Moreover, the drug showed antiangiogenic and proapoptotic actions, as revealed by vessel count and apoptotic index by terminal deoxynucleotide transferase-mediated nick end labeling in explanted tumors. Finally, treatment with indinavir did not block the production of vascular endothelial growth factor in the tumors. CONCLUSION: Indinavir could be helpful to prevent the development of hepatocarcinomas in HIV/HCV-coinfected individuals. In view of the current trend to substitute protease inhibitors with other antiretroviral agents, this information may have clinical implications.

Piroxicam and cisplatin in a mouse model of peritoneal mesothelioma.

PURPOSE: The aim of the present study was to evaluate the effects of piroxicam, a widely used nonsteroidal anti-inflammatory drug, alone and in combination with cisplatin (CDDP), on cell growth of mesothelioma cells. EXPERIMENTAL DESIGN: Cell proliferation, cell cycle analysis, and microarray technology were done on MSTO-211H and NCI-H2452 cells treated with piroxicam. Moreover, the effects of piroxicam and CDDP on tumor growth and survival of mouse xenograft models of mesothelioma were determined. RESULTS: Piroxicam treatment of MSTO-211H and NCI-H2452 cells resulted in a significant inhibition of proliferation. Cell cycle analysis revealed that there was an increase in the rate of apoptosis in MSTO-211H cells and an increase in the cells accumulating in G2-M in NCI-H2452. Moreover, a marked tumor growth inhibition and an extended survival of mice treated with a combination of piroxicam and CDDP in MSTO-211H cell-induced peritoneal mesotheliomas was observed. Last, GeneChip array analysis of MSTO-211H mesothelioma cell line revealed that piroxicam treatment caused up-regulation of metabolic pathway-associated genes and down-regulation of genes related to RNA processing apparatus. Of note, epidermal growth factor receptor, one of the new biological targets of chemotherapy for mesothelioma, was down-regulated and HtrA1, a serine protease recently shown to be an endogenous mediator of CDDP cytotoxicity, was up-regulated following piroxicam treatment both in vitro and in vivo. CONCLUSION: These data suggest that piroxicam sensitizes mesothelioma cells to CDDP-induced cytotoxicity by modulating the expression of several target genes. Therefore, piroxicam in combination with CDDP might potentially be useful in the treatment of patients with mesothelioma.

Erratum to: Safety Considerations of Inhaled Corticosteroids in the Elderly.

Inhaled corticosteroids (ICSs) are widely used in the treatment of patients with chronic obstructive pulmonary diseases. However, high-dose regimens and long-term use of ICSs have the potential to cause a variety of local and systemic side effects such as candidiasis, cataracts, glaucoma, and osteoporosis. The use of ICSs can also be associated with the risk of bone fractures, diabetes mellitus and pneumonia. These ICS-related side effects are of particular importance in elderly patients due to the presence of comorbidities and age-related behavioral, cognitive, and psychological problems, which can all interact with inhaled treatment. We reviewed the available literature on the clinically relevant side effects of ICSs in the elderly to provide practical measures to properly monitor and manage the risk of ICSs in the geriatric population. Inspection of the mouth, monitoring of ocular pressure, and use of bone-protective drugs may be necessary in patients on prolonged ICS therapy. Above all, the use of the lowest possible ICS dose and a careful re-assessment of the inhalation procedure should be recommended. Taken together, these observations suggest that physicians should use ICSs appropriately for those patients in whom the benefit will outweigh the risk, especially chronic obstructive pulmonary disease (COPD) patients with previous frequent exacerbations. Given the paucity of information on the topic and the need to extrapolate the results from studies with broader age ranges, we strongly encourage the design of specifically tailored clinical studies in the elderly.

Safety considerations of inhaled corticosteroids in the elderly.

Inhaled corticosteroids (ICSs) are widely used in the treatment of patients with chronic obstructive pulmonary diseases. However, high-dose regimens and long-term use of ICSs have the potential to cause a variety of local and systemic side effects such as candidiasis, cataracts, glaucoma, and osteoporosis. The use of ICSs can also be associated with the risk of bone fractures, diabetes mellitus and pneumonia. These ICS-related side effects are of particular importance in elderly patients due to the presence of comorbidities and age-related behavioral, cognitive, and psychological problems, which can all interact with inhaled treatment. We reviewed the available literature on the clinically relevant side effects of ICSs in the elderly to provide practical measures to properly monitor and manage the risk of ICSs in the geriatric population. Inspection of the mouth, monitoring of ocular pressure, and use of bone-protective drugs may be necessary in patients on prolonged ICS therapy. Above all, the use of the lowest possible ICS dose and a careful re-assessment of the inhalation procedure should be recommended. Taken together, these observations suggest that physicians should use ICSs appropriately for those patients in whom the benefit will outweigh the risk, especially chronic obstructive pulmonary disease (COPD) patients with previous frequent exacerbations. Given the paucity of information on the topic and the need to extrapolate the results from studies with broader age ranges, we strongly encourage the design of specifically tailored clinical studies in the elderly.

Electroporation as a strategy to promote HtrA1 gene uptake and chemotherapy efficacy in a mouse model of mesothelioma.

There is not a consensus on the best therapeutic approach to mesothelioma and the prognosis is still dismal. We have recently demonstrated that HtrA1 is a potential therapeutic target in mesothelioma cells. In this manuscript we describe that electroporation in a mouse mesothelioma xenograft was able to facilitate the expression of exogenous HtrA1 injected intra-lesionally in the tumors and to increase the penetration in the neoplastic cells of cisplatin given intra-peritoneally. Indeed, HtrA1 over-expression caused a significant slowing down of tumor growth; moreover, cisplatin efficacy in reducing tumor mass was amplified by electroporation and this phenomenon was even more significant when combining the electroporation of cisplatin and HtrA1. Considering that a substantial number of mesothelioma patients develop early local recurrence, even with radical resection combined with aggressive chemo- and radiotherapy, this multi-modality approach could be very effective in improving local tumor control after surgery. The identification of effective combination coupled with the development of novel equipments and electrodes will be instrumental in planning the translation of these results to humans as per correct laboratory-clinical interface.

Effects of exercise training on airway closure in asthmatics.

We previously reported that responsiveness to methacholine (Mch) in the absence of deep inspiration (DI) decreased in healthy subjects after a short course of exercise training. We assessed whether a similar beneficial effect of exercise on airway responsiveness could occur in asthmatics. Nine patients (male/female: 3/6; mean age ± SD: 24 ± 2 yr) with mild untreated asthma [forced expiratory volume in 1 s (FEV(1)): 100 ± 7.4% pred; FEV(1)/vital capacity (VC): 90 ± 6.5%] underwent a series of single-dose Mch bronchoprovocations in the absence of DI in the course of a 10-wk training rowing program (6 h/wk of submaximal and maximal exercise), at baseline (week 0), and at week 5 and 10. The single-dose Mch was established as the dose able to induce ≥ 15% reduction in inspiratory vital capacity (IVC) and was administered to each subject at every challenge occasion. Five asthmatics (male/female: 1/4; mean age ± SD: 26 ± 3 yr) with similar baseline lung function (FEV(1): 102 ± 7.0% predicted; FEV(1)/VC: 83 ± 6.0%; P = 0.57 and P = 0.06, respectively) not participating in the exercise training program served as controls. In the trained group, the Mch-induced reduction in IVC from baseline was 22 ± 10% at week 0, 13 ± 11% at week 5 (P = 0.03), and 11 ± 8% at week 10 (P = 0.028). The Mch-induced reduction in FEV(1) did not change with exercise (P = 0.69). The reduction in responsiveness induced by exercise was of the same magnitude of that previously obtained in healthy subjects (50% with respect to pretraining). Conversely, Mch-induced reduction in IVC in controls remained unchanged after 10 wk (%reduction IVC at baseline: 21 ± 20%; after 10 wk: 29 ± 14%; P = 0.28). This study indicates that a short course of physical training is capable of reducing airway responsiveness in mild asthmatics.

Apoptosis induced by piroxicam plus cisplatin combined treatment is triggered by p21 in mesothelioma.

BACKGROUND: Malignant mesothelioma (MM) is a rare, highly aggressive tumor, associated to asbestos exposure. To date no chemotherapy regimen for MM has proven to be definitively curative, and new therapies for MM treatment need to be developed. We have previously shown in vivo that piroxicam/cisplatin combined treatment in MM, specifically acts on cell cycle regulation triggering apoptosis, with survival increase. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed, at molecular level, the apoptotic increase caused by piroxicam/cisplatin treatment in MM cell lines. By means of genome wide analyses, we analyzed transcriptional gene deregulation both after the single piroxicam or cisplatin and the combined treatment. Here we show that apoptotic increase following combined treatment is mediated by p21, since apoptotic increase in piroxicam/cisplatin combined treatment is abolished upon p21 silencing. CONCLUSIONS/SIGNIFICANCE: Piroxicam/cisplatin combined treatment determines an apoptosis increase in MM cells, which is dependent on the p21 expression. The results provided suggest that piroxicam/cisplatin combination might be tested in clinical settings in tumor specimens that express p21.

Biological agents involved in malignant mesothelioma: relevance as biomarkers or therapeutic targets.

Malignant mesothelioma (MM) is a rare, highly aggressive tumor that arises from the surface serosal cells (pleural, peritoneal and pericardial cavities). Epidemiological and clinical data show that there is an association between asbestos exposure and MM development, even if the exact mechanism whereby asbestos induces MM is unknown. The continuing identification and elucidation of the molecular defects involved in mesothelioma pathogenesis and progression should lead to better disease control and greater therapeutic options in the near future. Goal of this article is to summarize the most recent advances in molecular pathogenesis of mesothelioma with particular emphasis on genes that could be considered as biomarkers or therapeutic targets and discuss possible clinical implications of these findings.

Expression of the embryonic lethal abnormal vision-like protein HuR in human mesothelioma: association with cyclooxygenase-2 and prognosis.

BACKGROUND: The human embryonic lethal abnormal vision (ELAV)-like protein HuR is a messenger RNA (mRNA)-binding protein that controls the stability of certain transcripts, including cyclooxygenase2 (COX-2). METHODS: To investigate a possible contribution of dysregulation of mRNA stability to the progression of cancer and to COX-2 over expression in mesothelioma, the authors studied expression of COX-2 and HuR in 5 mesothelioma cell lines (MSTO, NCI, Ist-Mes1, Ist-Mes2, and MPP89) and in a group of 29 human mesothelioma specimens that were characterized previously for COX-2 expression. RESULTS: All 5 cell lines expressed HuR, whereas COX-2 was not detectable in MSTO or NCI cells. Treatment with cytokines induced a shift in systolic HuR protein levels in MPP89 and Ist-Mes2 cells that was accompanied by an increase in the expression of COX-2 mRNA and protein. In Ist-Mes1 cells, cytokine stimulation did not cause the passage of HuR from nucleus to cytoplasm, and the synthesis of COX-2 did not increase. In tumor tissues, immunohistochemistry revealed a positive, statistically significant correlation between high COX-2 expression and cytoplasmic localization of HuR (P = .016). Moreover, on univariate analysis, overall survival was found to be influenced strongly by cytoplasmic HuR localization (P = .004). CONCLUSIONS: The current results suggested that HuR plays a role in tumor progression in mesothelioma and that COX-2 may be a target of its activity in neoplastic cells. Together, these observations indicate that strategies aiming toward the modulation of HuR may have a potential clinical benefit in mesothelioma.

Survival versus apoptotic 17beta-estradiol effect: role of ER alpha and ER beta activated non-genomic signaling.

The capability of 17beta-estradiol (E2) to induce the non-genomic activities of its receptors (ER alpha and ER beta) and to evoke different signaling pathways committed to the regulation of cell proliferation has been analyzed in different cell cancer lines containing transfected (HeLa) or endogenous (HepG2, DLD1) ER alpha or ER beta. In these cell lines, E2 induced different effects on cell growth/apoptosis in dependence of ER isoforms present. The E2-ER alpha complex rapidly activated multiple signal transduction pathways (i.e., ERK/MAPK, PI3K/AKT) committed to both cell cycle progression and apoptotic cascade prevention. On the other hand, the E2-ER beta complex induced the rapid and persistent phosphorylation of p38/MAPK which, in turn, was involved in caspase-3 activation and cleavage of poly(ADP-ribose)polymerase, driving cells into the apoptotic cycle. In addition, the E2-ER beta complex did not activate any of the E2-ER alpha-activated signal molecules involved in cell growth. Taken together, these results demonstrate the ability of ER beta isoform to activate specific signal transduction pathways starting from plasma membrane that may justify the effect of E2 in inducing cell proliferation or apoptosis in cancer cells. In particular this hormone promotes cell survival through ER alpha non-genomic signaling and cell death through ER beta non-genomic signaling.

Mechanisms of naringenin-induced apoptotic cascade in cancer cells: involvement of estrogen receptor alpha and beta signalling.

The flavanone naringenin (Nar), especially abundant in the Mediterranean diet, is reported to have anti-proliferative effects in many cancer cell lines. Antioxidant activities, kinase and glucose uptake inhibition have been proposed as molecular mechanisms for these effects. In addition, an anti-estrogenic activity has been observed but, at the present, it is poorly understood whether this latter activity could play a role in the Nar anti-tumoral effects. Here, we tested the ability of Nar to activate a specific, rapid signal transduction pathway committed to the generation of an apoptotic cascade in the presence of one of the two estrogen receptor (ER) isoforms (i.e., ERalpha or ERbeta). Cancer cells containing transfected (human cervix epitheloid carcinoma HeLa cells) or endogenous ERalpha (human hepatoma HepG2 cells) or ERbeta (human colon adenocarcinoma DLD-1 cells) were used. Our results show that Nar exerts an anti-proliferative effect only in the presence of ERalpha or ERbeta. Moreover, Nar stimulation induces the activation of p38/MAPK leading to the pro-apoptotic caspase-3 activation and to the poly(ADP-ribose) polymerase cleavage in all cancer cell lines considered. Notably, Nar shows an anti-estrogenic effect only in ERalpha containing cells; whereas in ERbeta containing cells, Nar mimics the 17beta-estradiol effects. These findings indicate new steps in the mechanism underlying ER-dependent anti-proliferative effects of Nar suggesting new potential chemopreventive actions of flavonoids on cancer growth.