DHEA-S, Androstenedione, 17-ß-estradiol signature as novel biomarkers for early prediction of risk of malignant pleural mesothelioma linked to asbestos-exposure: A preliminary investigation.

17-ß-estradiol, involved in mesothelioma pathogenesis, and its precursors were explored as potential biomarkers for the early diagnosis of mesothelioma. Using enzyme-linked immunosorbent assay(ELISA) for 17-ß-estradiol and ultra-high performance liquid chromatography/tandem mass spectrometry(UHPLC-MS/MS) for 19 17-ß-estradiol precursors, a comprehensive analysis of 20steroid hormones was conducted in the serum of mesothelioma patients(n=67), asbestos-exposed healthy subjects(n=39), and non-asbestos-exposed healthy subjects(n=35). Bioinformatics analysis explored three potential serum biomarkers: 17-ß-estradiol, DHEA-S, and androstenedione. The results revealed significant differences in 17-ß-estradiol levels between mesothelioma patients and both non-asbestos-exposed and asbestos-exposed healthy subjects. No significant variations in serum 17-ß-estradiol levels were observed among mesothelioma patients at different stages, suggesting its potential as an early diagnostic marker. 17-ß-estradiol levels were similar in mesothelioma patients with environmental and occupational asbestos exposure, while males with occupational asbestos exposure exhibited significantly higher levels of 17-ß-estradiol compared to females. Significant reduction in androstenedione and an increase in DHEA-S were observed in asbestos-exposed individuals compared to non-asbestos-exposed individuals. The analysis of DHEA-S-androstenedione-17-ß-estradiol signature score showed an increase in asbestos-exposed individuals and mesothelioma patients compared to non-asbestos-exposed individuals, and this score effectively distinguished between the groups. The Cancer Genome Atlas data was utilized to analyze the expression of 5-α-reductase1 and hydroxysteroid-17ß-dehydrogenase2 genes. The findings indicated that mesothelioma patients with elevated gene values for 5-α-reductase1 and hydroxysteroid-17ß-dehydrogenase2 have a worse or better prognosis on overall survival, respectively. In conclusion, this study suggests 17-ß-estradiol, DHEA-S, and androstenedione as biomarkers for mesothelioma risk and early diagnosis of mesothelioma in asbestos-exposed individuals, aiding timely intervention and improved care.

Identification of novel COX-2 / CYP19A1 axis involved in the mesothelioma pathogenesis opens new therapeutic opportunities.

BACKGROUND: Based on previous studies highlighting that the induction of cyclooxygenase-2 (COX-2) and high prostaglandin E2 (PGE2) levels contribute to the pathogenesis of malignant pleural mesothelioma (MPM), and that aromatase (CYP19A1), an enzyme that plays a key role in estrogen biosynthesis, along with estradiol (E2) were expressed in MPM, this study aimed to investigate the possible interplay between COX-2 and CYP19A1 in the pathogenesis of mesothelioma, as well as the underlying mechanism. METHODS: The interaction between COX-2 and CYP19A1 was first investigated on different MPM lines upon PGE2, and COX-2 inhibitor (rofecoxib) treatment by western blot, RT-PCR. The key regulatory pathways involved in the COX-2 and CYP19A1 axis were further studied in MPM cells, after rofecoxib and exemestane (CYP19A1 inhibitor) treatment in monotherapy and in combination, by cell cycle distribution, western blot, and combination index analysis. To explore the role of COX-2/CYP19A1 axis in 3D preclinical models of MPM cells, we analyzed the effect of combination of COX-2 and CYP19A1 inhibitors in mesosphere formation. Immunohistochemical analysis of MPM mesosphere and specimens was utilized to evaluate the involvement of COX-2 on the CYP19A1 activity and the relationship between E2 and COX-2. RESULTS: PGE2 or rofecoxib treatment caused in MPM cells an increased or decreased, respectively, CYP19A1 expression at mRNA and protein levels. The effect of rofecoxib and exemestane combination in MPM cell proliferation was synergistic. Activation of caspase-3 and cleavage of PARP confirmed an apoptotic death for MPM cell lines. Increased expression levels of p53, p21, and p27, downregulation of cyclin D1 and inhibition of Akt activation (pAKT) were also found. The antagonistic effect of rofecoxib and exemestane combination found only in one cell line, was reverted by pretreatment with MK2206, a pAKT inhibitor, indicating pAKT as an actionable mediator in the COX-2-CYP19A1 axis. Reduction of size and sphere-forming efficiency in MPM spheres after treatment with both inhibitor and a decrease in COX-2 and E2 staining was found. Moreover, immunohistochemical analysis of 46 MPM samples showed a significant positive correlation between COX-2 and E2. CONCLUSIONS: Collectively, the results highlighted a novel COX-2/CYP19A1 axis in the pathogenesis of MPM that can be pharmacologically targeted, consequently opening up new therapeutic options.

The effect of CELLFOODTM on radiotherapy or combined chemoradiotherapy: preclinical evidence.

BACKGROUND: Based on previous observations that the nutraceutical CELLFOOD™ (CF), the 'physiological modulator' that aimed to make oxygen available 'on demand', inhibits the growth of cancer cells, this study was designed to investigate the role of CF in the regulation of hypoxia-inducible factor 1 alpha (HIF1α) and its correlated proteins, phosphoglycerate kinase 1 and vascular endothelial growth factor. Our idea was that CF, acting on HIF1α, in combination with current anticancer therapies could improve their effectiveness. METHODS: To evaluate the effect of CF in association with radiotherapy and chemotherapy, different human cancer cell lines and mice with mesothelioma were analysed by tumour growth, clonogenic assay, western blot and immunohistochemical analysis. RESULTS: CF in combination with radiation with or without cisplatin increases the death rate of cancer cells. In vivo, 70% of mice treated with CF before the mesothelioma graft did not show any tumour growth, indicating a possible preventive effect of CF. Moreover, in mouse mesothelioma xenografts, CF improves the effect of radiotherapy also in combination with chemotherapy treatment. Immunohistochemical analysis of tumour explants showed that HIF1α expression was reduced by the combination of CF and radiotherapy treatment and even more by the combination of CF and radiotherapy and chemotherapy treatment. Mechanistically, CF increases the fraction of oxygenated cells, making the radiotherapy more effective with a greater production of reactive oxygen species (ROS) that in turn, reduce the HIF1α expression. This effect is amplified by further increase in ROS from chemotherapy. CONCLUSIONS: Collectively, results from preclinical trials suggest that CF could be a useful intervention to improve the efficacy of radiotherapy or combined treatment strategies and could be a promising treatment modality to counteract cancer.

HSP90 inhibition alters the chemotherapy-driven rearrangement of the oncogenic secretome.

Adaptive resistance to therapy is a hallmark of cancer progression. To date, it is not entirely clear how microenvironmental stimuli would mediate emergence of therapy-resistant cell subpopulations, although a rearrangement of the cancer cell secretome following therapy-induced stress can be pivotal for such a process. Here, by using the highly chemoresistant malignant pleural mesothelioma (MPM) as an experimental model, we unveiled a key contribution of the chaperone HSP90 at assisting a chemotherapy-instigated Senescence-Associated-Secretory-Phenotype (SASP). Thus, administration of a clinical trial grade, HSP90, inhibitor blunted the release of several cytokines by the chemotherapy-treated MPM cells, including interleukin (IL)-8. Reduction of IL-8 levels hampered the FAK-AKT signaling and inhibited 3D growth and migration. This correlated with downregulation of key EMT and chemoresistance genes and affected the survival of chemoresistant ALDHbright cell subpopulations. Altogether, inhibition of HSP90 provoked a switch from a pro-tumorigenic SASP to a pro-apoptotic senescence status, thus resulting in chemosensitizing effects. In mouse xenografts treated with first-line agents, inhibiting HSP90 blunted FAK activation and reduced the expression of ALDH1A3 and the levels of circulating human IL-8, these latter strongly correlating with the effect on tumor growth. We validated the above findings in primary mesothelioma cultures, a more clinically relevant model. We unveiled here a key contribution of the chaperone HSP90 at assisting the secretory stress in chemotherapy-treated cells, which may warrant further investigation in combinatorial therapeutic settings.

Modulation of reactive oxygen species via ERK and STAT3 dependent signalling are involved in the response of mesothelioma cells to exemestane.

Pleural mesothelioma is a deadly form of cancer. The prognosis is extremely poor due to the limited treatment modalities. Uptake of asbestos fibres, the leading cause of mesothelioma, lead to the accumulation of reactive-oxygen-species (ROS). Interestingly, increasing ROS production by using ROS-generating drugs may offer a strategy to selectively trigger cell death. Exemestane, an aromatase inhibitor, has previously shown anti-tumor properties in mesothelioma preclinical models suggesting a role of G protein-coupled receptor 30 (GPR30) in the drug response. As exemestane, in addition to blocking estrogen biosynthesis, generates ROS that are able to arrest the growth of breast cancer, we explored the role of ROS, antioxidant defense system, and ROS-induced signalling pathways in mesothelioma cells during exemestane response. Here we report that exemestane treatment reduced cell proliferation with an increase in ROS production and reduction of cyclic adenosine monophosphate (cAMP) levels in MSTO-H211, Ist-Mes1, Ist-Mes2 and MPP89 exemestane-sensitive mesothelioma cell lines, but not in NCI-H2452 exemestane-insensitive mesothelioma cells. Exemestane induced a significant antioxidant response in NCI-H2452 cells, as highlighted by an increase in γ-glutamylcysteine levels, catalase (Cat), superoxide-dismutase and (SOD) and glutathione-peroxidase (GSH-Px) activity and nuclear factor E2-related factor 2 (Nrf2) activation, responsible for drug insensitivity. Conversely, exemestane elevated ROS levels along with increased ERK phosphorylation and a reduction of p-STA3 in exemestane-sensitive mesothelioma cells. ROS generation was the crucial event of exemestane action because ROS inhibitor N-acetyl-L-cysteine (NAC) abrogated p-ERK and p-STAT3 modulation and cellular death. Exemestane also modulates ERK and STAT3 signalling via GPR30. Results indicate an essential role of ROS in the antiproliferative action of exemestane in mesothelioma cells. It is likely that the additional oxidative insults induced by exemestane results in the lethal effects of mesothelioma cells by increasing ROS production. As such, manipulating ROS levels with exemestane seems to be a feasible strategy to selectively kill mesothelioma cells with less toxicity to normal cells by regulating ERK and STAT3 activity.

Reduced cell viability and apoptosis induction in human thyroid carcinoma and mesothelioma cells exposed to cidofovir.

Besides its well-recognized antiviral activity, Cidofovir (CDV) has been shown to exert anticancer properties both within in vitro and in vivo models. The aim of this study was to evaluate the effects of CDV on still unexplored cultured cancer cells from human mesothelioma as well as breast, colon, liver, lung, prostate, and thyroid carcinomas. Overall, a dose- and time-dependent inhibition of cell viability was observed after CDV exposure. To clarify the mechanisms underlying CDV action, apoptotic cell death was investigated in two infected cell lines [Ist-Mes1 and Ist-Mes2 mesothelioma cells (SV40+)] and in two uninfected cell lines (NCI-H2425 mesothelioma cells and FTC-133 thyroid cancer cells), which resulted the most sensitive to CDV treatment. Reduced expression of procaspase-3 and increased expression of PARP p85 fragment were observed in both infected and uninfected mesothelioma cells, indicating apoptosis induction by CDV in a virus-independent manner. Similarly, the increase of the pro-apoptotic proteins p53, cytochrome c and caspase-3, the decrease of the survival protein Bcl-x, and the increment of Bax/Bcl-2 ratio revealed the occurrence of apoptosis in CDV-treated FTC-133. The presence of nuclear DNA fragmentation confirmed apoptotic cell death by CDV. Overall, our findings warrant further investigations to explore the therapeutic potential of CDV for human mesothelioma and follicular thyroid carcinoma.

Reduction of estradiol in human malignant pleural mesothelioma tissues may prevent tumour growth, as implied by in in-vivo and in-vitro models.

This study aimed to investigate intratumoural estradiol and estrogen-receptors (ERα, ERß and GPR30) in malignant pleural mesothelioma (MPM) to understand their function. Here, we report that immunohistochemistry of estradiol showed cytoplasmatic staining in 95% of fifty-seven human MPM samples with a trend toward a negative correlation between estradiol levels and the median post-diagnosis survival time. ERß was only focally positive in 5.3% of cases, GPR30 and ERα were negative in our cases of MPM. GPR30 was detected mainly in glycosylated form in MPM cells. Moreover, G15, a GPR30 antagonist, induced MPM cell death. Altogether, these data suggest that MPM cells produce E2 interact with glycosylated forms of GPR30, and this facilitates tumour growth. Estradiol was found in MPM cells and plasma from mice mesothelioma xenografts. Concurrent reduction in tumour mass and plasmatic estradiol levels were observed in the mice treated with exemestane, suggesting that the reduction of E2 levels inhibit MPM growth. Thus, it appears that agents reducing estradiol levels could be useful to MPM therapy.

Reactive oxygen species a double-edged sword for mesothelioma.

It is well known that oxidative stress can lead to chronic inflammation which, in turn, could mediate most chronic diseases including cancer. Oxidants have been implicated in the activity of crocidolite and amosite, the most powerful types of asbestos associated to the occurrence of mesothelioma. Currently rates of mesothelioma are rising and estimates indicate that the incidence of mesothelioma will peak within the next 10-15 years in the western world, while in Japan the peak is predicted not to occur until 40 years from now. Although the use of asbestos has been banned in many countries around the world, production of and the potentially hazardous exposure to asbestos is still present with locally high incidences of mesothelioma. Today a new man-made material, carbon nanotubes, has arisen as a concern; carbon nanotubes may display 'asbestos-like' pathogenicity with mesothelioma induction potential. Carbon nanotubes resulted in the greatest reactive oxygen species generation. How oxidative stress activates inflammatory pathways leading to the transformation of a normal cell to a tumor cell, to tumor cell survival, proliferation, invasion, angiogenesis, chemoresistance, and radioresistance, is the aim of this review.

Exemestane blocks mesothelioma growth through downregulation of cAMP, pCREB and CD44 implicating new treatment option in patients affected by this disease.

BACKGROUND: Recent evidence suggests that aromatase may be involved in the pathogenesis of malignant mesothelioma. Here, we evaluated the effect of exemestane, an inhibitor of aromatase, in the treatment of mesothelioma using in vitro and in vivo preclinical models. RESULTS: We show a significant reduction of cell proliferation, survival, migration and block of cells in S phase of cell cycle in mesothelioma cells upon exemestane treatment. Moreover, we find that CD44, which is involved in mesothelioma cells migration, was modulated by exemestane via cAMP and pCREB. Most importantly, in mice mesothelioma xenograft exemestane causes a significant decrease in tumor size and the association pemetrexed/exemestane is more effective than pemetrexed/cisplatin. CONCLUSION: The preclinical mesothelioma model suggests that exemestane might be beneficial in mesothelioma treatment.

CELLFOOD™ induces apoptosis in human mesothelioma and colorectal cancer cells by modulating p53, c-myc and pAkt signaling pathways.

BACKGROUND: CELLFOOD™ (CF) is a nutraceutical non-addictive, non-invasive, and completely non-toxic unique proprietary colloidal-ionic formula. Little is known about its effect on cancer cells in solid tumors. The aim of this study was to evaluate the effect that CF has on different cancer cell lines and the mechanism by which the nutraceutical works. METHODS: The effect of CF on HFF (normal fibroblasts), Met5A (mesothelium), MSTO-211H, NCI-2452, Ist-Mes1, MPP89, Ist-Mes2 (mesothelioma), M14 (melanoma), H1650, H1975 (lung cancer), SKRB3 (breast cancer), and HCT-116 (colorectal cancer) cell growth was tested by cell proliferation and clonogenic assay. Among all of them, MSTO-211 and HCT-116 were analyzed for cell cycle by flow cytometry and western blot. RESULTS: All human cancer lines were suppressed on cell growth upon 1:200 CF treatment for 24 and 48 hours. Death was not observed in HFF and Met5A cell lines. Cell cycle analysis showed an increased sub-G1 with reduction of G1 in MSTO-211 and a cell cycle arrest of in G1 in HCT116. Activation of caspase-3 and cleavage of PARP confirmed an apoptotic death for both cell lines. Increased expression levels of p53, p21, and p27, downregulation of c-myc and Bcl-2, and inhibition of Akt activation were also found in CF-treated MSTO-211 and HCT-116 cells. CONCLUSIONS: These findings ascertained an interaction between p53, c-myc, p21, p27, Bcl-2, PI3K/Akt pathway, and CF-induced apoptosis in MSTO-211H and HCT-116 cells, suggesting that CF acts as an important regulator of cell growth in human cancer cell lines. CF could be a useful nutraceutical intervention for prevention in colon cancer and mesothelioma.

Metabolism modifications and apoptosis induction after Cellfood™ administration to leukemia cell lines.

BACKGROUND: Cellfood™ (CF) is a nutritional supplement containing deuterium sulphate, minerals, amino acids, and enzymes, with well documented antioxidant properties. Its organic and inorganic components are extracted from the red algae Lithothamnion calcareum, whose mineral extract has shown growth-inhibitory effect both on in vitro and in vivo models. The purpose of this study was to evaluate the antiproliferative effects of CF on leukemic cells. In fact, according to its capacity to modulate O2 availability and to improve mitochondrial respiratory metabolism, we wondered if CF could affect cancer cell metabolism making cells susceptible to apoptosis. METHODS: Three leukemic cell lines, Jurkat, U937, and K562, were treated with CF 5 µl/ml up to 72 hours. Cell viability, apoptosis (i.e. caspase-3 activity and DNA fragmentation), hypoxia inducible factor 1 alpha (HIF-1α) concentration, glucose transporter 1 (GLUT-1) expression, lactate dehydrogenase (LDH) activity and lactate release in the culture medium were detected and compared with untreated cells. RESULTS: CF significantly inhibited leukemic cell viability by promoting cell apoptosis, as revealed by caspase-3 activation and DNA laddering. In particular, CF treated cells showed lower HIF-1α levels and lower GLUT-1 expression as compared to untreated cells. At the same time, CF was able to reduce LDH activity and, consequently, the amount of lactate released in the extracellular environment. CONCLUSIONS: We supplied evidence for an antiproliferative effect of CF on leukemia cell lines by inducing cell death through an apoptotic mechanism and by altering cancer cell metabolism through HIF-1α and GLUT-1 regulation. Thanks to its antioxidative and proapoptotic properties, CF might be a good candidate for cancer prevention.

Cyclooxygenase-2, epidermal growth factor receptor, and aromatase signaling in inflammation and mesothelioma.

Malignant mesothelioma or mesothelioma is a rare form of cancer that develops from transformed cells originating in the mesothelium, the protective lining that covers many of the internal organs of the body. It is directly linked to asbestos exposure, which acts as a carcinogen by initiating the carcinogenic process. Because of their shape, asbestos fibers can cross the membrane barriers inside the body and cause inflammatory and fibrotic reactions. Such reactions are believed to be the mechanism by which asbestos fibers may trigger malignant mesothelioma in the pleural membrane around the lungs. Carcinogens are known to modulate the transcription factors, antiapoptotic proteins, proapoptotic proteins, protein kinases, cell-cycle proteins, cell adhesion molecules, COX-2, and growth factor signaling pathways. This article reviews recent studies regarding some malignant mesothelioma molecular targets not only for cancer prevention but also for cancer therapy.

Pharmacokinetics of gemcitabine at fixed-dose rate infusion in patients with normal and impaired hepatic function.

BACKGROUND AND OBJECTIVES: Gemcitabine (2,2-difluorodeoxycytidine [dFdC]) can be administered in a standard 30-minute infusion or in a fixed-dose-rate (FDR) infusion to maximize the rate of accumulation of triphosphate, its major intracellular metabolite. The standard 30-minute infusion requires dose adjustment in patients with organ dysfunction, especially in patients with elevated baseline serum bilirubin levels. On the other hand, the FDR infusion is burdened by increased haematological toxicity. The primary aim of this study was to evaluate the pharmacokinetics of dFdC and its metabolite difluorodeoxyuridine (dFdU) in patients with normal and impaired hepatic function. PATIENTS AND METHODS: In this prospective study, patients with pancreatic or biliary tract carcinoma and normal or impaired hepatic function tests were considered eligible for recruitment. Patients were recruited according to the following criteria: (i) serum bilirubin <1.6 mg/dL and AST and ALT <2 times the upper the limit of normal (ULN) [cohort I]; and (ii) serum bilirubin >1.6 mg/dL and/or AST/ALT >2 times the ULN (cohort II). An FDR infusion of gemcitabine 1000 mg/m2 was administered on days 1, 8 and 15 every 4 weeks. The pharmacokinetic analysis of gemcitabine and dFdU was performed with high-performance liquid chromatography-tandem mass spectrometry assay in cycles 1 and 2. RESULTS: Thirteen patients were enrolled, four in cohort I and nine in cohort II. All patients were assessable for toxicity and pharmacokinetic analysis. The grade and rate of toxicities were similar in both groups, and patients with elevation of bilirubin and/or transaminases did not require dose reduction of gemcitabine. Pharmacokinetic analysis revealed a reduction of the experimental area under the plasma concentration-time curve for gemcitabine and dFdU in patients with hepatic dysfunction when compared with patients with normal hepatic function. All other pharmacokinetic parameters were similar in the two cohorts. No statistical difference was demonstrated for all parameters evaluated between cycle 1 and cycle 2 in the two groups. CONCLUSION: Gemcitabine 1000 mg/m2 can be administered as an FDR infusion in patients with altered hepatic function without causing additional toxicity compared with patients with normal hepatic function.